Pyrazolo compounds and uses thereof

ABSTRACT

The present invention relates to compounds useful as inhibitors of one or more histone demethylses. The invention also provides pharmaceutically acceptable compositions comprising compounds of the present invention and methods of using said compositions in the treatment of various disorders.

PRIORITY OF INVENTION

This application claims priority from U.S. Provisional Application No. 61/778,759, filed 13 Mar. 2013. The entire content of this provisional application is hereby incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds useful as inhibitors of histone demethylases.

BACKGROUND OF THE INVENTION

Packaging the 3 billion nucleotides of the human genome into the nucleus of a cell requires tremendous compaction. To accomplish this feat, DNA in our chromosomes is wrapped around spools of proteins called histones to form dense repeating protein/DNA polymers known as chromatin: the defining template for gene regulation. Far from serving as mere packaging modules, chromatin templates form the basis of a newly appreciated and fundamentally important set of gene control mechanisms termed epigenetic regulation. By conferring a wide range of specific chemical modifications to histones and DNA, epigenetic regulators modulate the structure, function, and accessibility of our genome, thereby exerting a tremendous impact on gene expression. Hundreds of epigenetic effectors have recently been identified, many of which are chromatin-binding or chromatin-modifying enzymes. Significantly, an increasing number of these enzymes have been associated with a variety of disorders such as neurodegenerative disorders, metabolic diseases, inflammation, and cancer. Thus, therapeutic agents directed against this emerging class of gene regulatory enzymes promise new approaches to the treatment of human diseases.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, and pharmaceutically acceptable compositions thereof, are effective as inhibitors of histone demethylases, including 2-oxoglutarate dependent enzymes such as Jumonji domain containing proteins, members of the H3K4 (histone 3 K4) demethylase family of proteins, and/or members of the JARID subfamily of histone demethylases. Such compounds are of formula I:

or a pharmaceutically acceptable salt thereof, wherein R¹ and Ring A are as defined and described herein.

Provided compounds, and pharmaceutically acceptable compositions thereof, are useful for treating a variety of diseases, disorders or conditions associated with abnormal cellular responses triggered by events mediated by histone demethylases such as 2-oxoglutarate dependent enzymes, Jumonji domain containing proteins, members of the H3K4 (histone 3K4) demethylase family of proteins, and/or members of the JARID subfamily of enzymes. Such diseases, disorders, or conditions include those described herein.

Provided compounds are also useful for the study of histone demethylases, such as 2-oxoglutarate dependent enzymes, Jumonji domain containing proteins, members of the H3K4 (histone 3 K4) demethylase family of proteins, and/or members of the JARID subfamily of enzymes in biological and pathological phenomena, the study of intracellular signal transduction pathways mediated by such histone demethylases, and the comparative evaluation of new inhibitors of these and other histone demethylases.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS 1. General Description of Compounds of the Invention

In certain embodiments, the present invention provides a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   R¹ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R,     —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂,     —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂,     —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂,     —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; -   each R is independently hydrogen or an optionally substituted group     selected from C₁₋₆ aliphatic, phenyl, a 3-7 membered saturated or     partially unsaturated carbocyclic ring, an 8-10 membered bicyclic     saturated, partially unsaturated or aryl ring, a 5-6 membered     monocyclic heteroaryl ring having 1-3 heteroatoms independently     selected from nitrogen, oxygen, or sulfur, a 4-7 membered saturated     or partially unsaturated heterocyclic ring having 1-2 heteroatoms     independently selected from nitrogen, oxygen, or sulfur, a 7-10     membered bicyclic saturated or partially unsaturated heterocyclic     ring having 1-4 heteroatoms independently selected from nitrogen,     oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring     having 1-4 heteroatoms independently selected from nitrogen, oxygen,     or sulfur; -   each R′ is independently —R, —C(O)R, —CO₂R, or two R′ on the same     nitrogen are taken together with their intervening atoms to form a     4-7 membered heterocyclic ring having 1-2 heteroatoms independently     selected from nitrogen, oxygen, and sulfur; -   Ring A is

-   R² and R³ are independently —R, halogen, —OR, —SR, —N(R′)₂, —CN,     —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R,     —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂,     —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂,     —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR,     —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or:     -   R² and R³ are taken together with their intervening atoms to         form an optionally substituted 5-7 membered partially         unsaturated or aromatic fused ring having 0-4 heteroatoms         independently selected from nitrogen, oxygen, and sulfur; -   R^(2′) is —R, —OR, —SR, —N(R′)₂, —C(O)R, —CO₂R, —C(O)N(R′)₂,     —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R,     —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R,     —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂,     —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or:     -   R^(2′) and R³ are taken together with their intervening atoms to         form an optionally substituted 5-7 membered partially         unsaturated or aromatic fused ring having 1-4 heteroatoms         independently selected from nitrogen, oxygen, and sulfur; -   X is —N(R⁴)—, —O—, or —S—; -   R⁴ is —R, —C(O)R, —CO₂R, or —S(O)₂R; or:     -   R⁴ and R³ are taken together with their intervening atoms to         form an optionally substituted 5-7 membered saturated, partially         unsaturated, or aromatic fused ring having 1-4 heteroatoms         independently selected from nitrogen, oxygen, and sulfur; -   R⁵ is R, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)C(O)R, or —C(O)CH₂C(O)R;     or:     -   R⁵ and R² are taken together with their intervening atoms to         form an optionally substituted 5-7 membered partially         unsaturated or aromatic fused ring having 1-4 heteroatoms         independently selected from nitrogen, oxygen, and sulfur; and -   R⁶ is —R, halogen, —OR, —SR, —N(R¹)₂, —CN, —NO₂, —C(O)R, —CO₂R,     —C(O)N(R¹)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂,     —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂,     —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂,     —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or:     -   R⁶ and R³ are taken together with their intervening atoms to         form an optionally substituted 5-7 membered partially         unsaturated or aromatic fused ring having 0-4 heteroatoms         independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, the present invention provides a compound of formula I other than any one of the following:

2. Compounds and Definitions

Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3^(rd) Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.

Where a particular enantiomer is preferred, it may, in some embodiments be provided substantially free of the corresponding enantiomer, and may also be referred to as “optically enriched.” “Optically-enriched,” as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain embodiments the compound is made up of at least about 90% by weight of a preferred enantiomer. In other embodiments the compound is made up of at least about 95%, 98%, or 99% by weight of a preferred enantiomer. Preferred enantiomers may be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972).

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as in N-substituted pyrrolidinyl)).

As used herein a “direct bond” or “covalent bond” refers to a single, double or triple bond. In certain embodiments, a “direct bond” or “covalent bond” refers to a single bond.

The terms “halo” and “halogen” as used herein refer to an atom selected from fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), and iodine (iodo, —I).

The term “aliphatic” or “aliphatic group”, as used herein, denotes a hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridging, and spiro-fused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-6 carbon atoms. In some embodiments, aliphatic groups contain 1-4 carbon atoms, and in yet other embodiments aliphatic groups contain 1-3 carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “unsaturated”, as used herein, means that a moiety has one or more units of unsaturation.

The terms “cycloaliphatic”, “carbocycle”, “carbocyclyl”, “carbocyclo”, or “carbocyclic”, used alone or as part of a larger moiety, refer to a saturated or partially unsaturated cyclic aliphatic monocyclic or bicyclic ring systems, as described herein, having from 3 to 10 members, wherein the aliphatic ring system is optionally substituted as defined above and described herein. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl. In some embodiments, the cycloalkyl has 3-6 carbons. The terms “cycloaliphatic”, “carbocycle”, “carbocyclyl”, “carbocyclo”, or “carbocyclic” also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl, tetrahydronaphthyl, decalin, or bicyclo[2.2.2]octane, where the radical or point of attachment is on an aliphatic ring.

As used herein, the term “cycloalkylene” refers to a bivalent cycloalkyl group. In certain embodiments, a cycloalkylene group is a 1,1-cycloalkylene group (i.e., a spiro-fused ring). Exemplary 1,1-cycloalkylene groups include

In other embodiments, a cycloalkylene group is a 1,2-cycloalkylene group or a 1,3-cycloalkylene group. Exemplary 1,2-cycloalkylene groups include

The term “alkyl,” as used herein, refers to a monovalent saturated, straight- or branched-chain hydrocarbon radical derived from an aliphatic moiety containing between one and six carbon atoms by removal of a single hydrogen atom. In some embodiments, alkyl contains 1-5 carbon atoms. In another embodiment, alkyl contains 1-4 carbon atoms. In still other embodiments, alkyl contains 1-3 carbon atoms. In yet another embodiment, alkyl contains 1-2 carbons. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like.

The term “alkenyl,” as used herein, denotes a monovalent group derived from a straight- or branched-chain aliphatic moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. In certain embodiments, alkenyl contains 2-6 carbon atoms. In certain embodiments, alkenyl contains 2-5 carbon atoms. In some embodiments, alkenyl contains 2-4 carbon atoms. In another embodiment, alkenyl contains 2-3 carbon atoms. Alkenyl groups include, for example, ethenyl (“vinyl”), propenyl (“allyl”), butenyl, 1-methyl-2-buten-1-yl, and the like.

The term “alkynyl,” as used herein, refers to a monovalent group derived from a straight- or branched-chain aliphatic moiety having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. In certain embodiments, alkynyl contains 2-6 carbon atoms. In certain embodiments, alkynyl contains 2-5 carbon atoms. In some embodiments, alkynyl contains 2-4 carbon atoms. In another embodiment, alkynyl contains 2-3 carbon atoms. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (“propargyl”), 1-propynyl, and the like.

The term “aryl” used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic and bicyclic ring systems having a total of five to 10 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term “aryl” may be used interchangeably with the term “aryl ring”. In certain embodiments of the present invention, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl”, as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenantriidinyl, or tetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar-”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 π electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be mono- or bicyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted. The term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.

As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 4- to 7-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or ⁺NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle”, “heterocyclyl”, “heterocyclyl ring”, “heterocyclic group”, “heterocyclic moiety”, and “heterocyclic radical”, are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, 2-azabicyclo[2.2.1]heptanyl, octahydroindolyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond between ring atoms but is not aromatic. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

The term “alkylene” refers to a bivalent alkyl group. An “alkylene chain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. Combinations of substituents envisioned under this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; —(CH₂)₀₋₄R^(o); —(CH₂)₀₋₄OR^(o); —O—(CH₂)₀₋₄C(O)OR^(o); —(CH₂)₀₋₄CH(OR^(o))₂; —(CH₂)₀₋₄SR^(o); —(CH₂)₀₋₄Ph, which may be substituted with R^(o); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substituted with R^(o); —CH═CHPh, which may be substituted with R^(o); —NO₂; —CN; —N₃; —(CH₂)₀₋₄N(R^(o))₂; —(CH₂)₀₋₄N(R^(o)C(O)R^(o); —N(R^(o))C(S)R^(o); —(CH₂)₀₋₄N(R^(o)C(O)NR^(o) ₂; —N(R^(o)C(S)NR^(o) ₂; —(CH₂)₀₋₄N(R^(o))C(O)OR^(o); —N(R^(o))N(R^(o)C(O)R^(o); —N(R^(o))N(R^(o)C(O)NR^(o) ₂; —N(R^(o))N(R^(o)C(O)OR^(o); —(CH₂)₀₋₄C(O)R^(o); —C(S)R^(o); —(CH₂)₀₋₄C(O)OR^(o); —(CH₂)₀₋₄C(O)SR^(o); —(CH₂)₀₋₄C(O)OSiR^(o) ₃; —(CH₂)₀₋₄OC(O)R^(o); —OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(o); —(CH₂)₀₋₄ SC(O)R^(o); —(CH₂)₀₋₄C(O)NR^(o) ₂; —C(S)NR^(o) ₂; —C(S)SR^(o); —SC(S)SR^(o), —(CH₂)₀₋₄OC(O)NR^(o) ₂; —C(O)N(OR^(o))R^(o); —C(O)C(O)R^(o); —C(O)CH₂C(O)R^(o); —C(NOR^(o))R^(o); —(CH₂)₀₋₄SSR^(o); —(CH₂)₀₋₄S(O)₂R^(o); —(CH₂)₀₋₄S(O)₂OR^(o); —(CH₂)₀₋₄OS(O)₂R^(o); —S(O)₂NR^(o) ₂; —(CH₂)₀₋₄S(O)R^(o); —N(R^(o))S(O)₂NR^(o) ₂; —N(R^(o)S(O)₂R^(o); —N(OR^(o))R^(o); —C(NH)NR^(o) ₂; —P(O)₂R^(o); —P(O)R^(o) ₂; —OP(O)R^(o) ₂; —OP(O)(OR^(o))₂; —SiR^(o) ₃; —(C₁₋₄ straight or branched)alkylene)O—N(R^(o))₂; or —(C₁₋₄ straight or branched)alkylene)C(O)O—N(R^(o))₂, wherein each R^(o) may be substituted as defined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R^(o), taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(o) (or the ring formed by taking two independent occurrences of R^(o) together with their intervening atoms), are independently halogen, —(CH₂)₀₋₂R^(•), -(haloR^(•)), —(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(•), —CH₂)₀₋₂CH(OR^(•))₂; —O(haloR^(•)), —CN, —N₃, —(CH₂)₀₋₂C(O)R^(•), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂SR^(•), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(•), —(CH₂)₀₋₂NR^(•) ₂, —NO₂, —SiR^(•) ₃, —OSiR^(•) ₃, —C(O)SR^(•), —(C₁₋₄ straight or branched alkylene)C(O)OR^(•), or —SSR^(•) wherein each R^(•) is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C₁₋₄ aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R^(o) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: ═O, ═S, ═NNR*₂, ═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or —S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selected from hydrogen, C₁₋₆ aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* is selected from hydrogen, C₁₋₆ aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen, —R^(•), -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —CN, —C(O)OH, —C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein each R^(•) is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†), —C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂, —C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†)S(O)₂R^(†); wherein each R^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substituted as defined below, unsubstituted —OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R^(†), taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independently halogen, —R^(•), -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —CN, —C(O)OH, —C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein each R^(•) is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

As used herein, the term “inhibitor” is defined as a compound that binds to and/or inhibits the target 2-oxoglutarated dependent enzyme with measurable affinity. In certain embodiments, an inhibitor has an IC₅₀ and/or binding constant of less about 50 μM, less than about 1 μM less than about 500 nM, less than about 100 nM, or less than about 10 nM.

The terms “measurable affinity” and “measurably inhibit,” as used herein, means a measurable change in activity of at least one 2-oxoglutarate dependent enzyme between a sample comprising a provided compound, or composition thereof, and at least one 2-oxoglutarate. dependent enzyme, and an equivalent sample comprising at least one 2-oxoglutarate dependent enzyme, in the absence of said compound, or composition thereof.

3. Description of Exemplary Compounds

In certain embodiments, the present invention provides a compound of formula I,

or a pharmaceutically acceptable salt thereof, wherein R¹ and Ring A are as defined and described herein.

As defined generally above, R¹ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂, wherein R and R′ are as defined above and described herein. In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is optionally substituted C₁₋₆ aliphatic. In certain embodiments, R¹ is optionally substituted C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl. In certain embodiments, R¹ is optionally substituted C₁₋₆ alkyl. In certain embodiments, R¹ is methyl. In certain other embodiments, R¹ is ethyl or tert-butyl. In some embodiments, R¹ is —OR, —SR, or —N(R′)₂. In certain embodiments, R¹ is —SR. In certain embodiments, R¹ is —NH₂. In certain embodiments, R¹ is —CN or —NO₂. In some embodiments, R¹ is halogen. In certain embodiments, R¹ is fluoro, chloro, bromo, or iodo. In certain embodiments, R¹ is fluoro. In some embodiments, R¹ is —C(O)R, —CO₂R, —C(O)SR, —C(O)N(R′)₂, —C(O)C(O)R, or —C(O)CH₂C(O)R. In certain embodiments, R¹ is —C(S)OR or —C(S)N(R′)₂. In other embodiments, R¹ is —S(O)R, —SO₂R, or —SO₂N(R′)₂. In some embodiments, R¹ is —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, or —N(R′)C(═N(R′))N(R′)₂. In certain embodiments, R¹ is —N(R′)N(R′)₂. In some embodiments, R¹ is —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂.

As defined generally above, Ring A is

wherein X, R², R^(2′), R³, R⁵, and R⁶ are as defined above and described herein. Thus, in certain embodiments, a compound of the invention is of one of the following formulae:

wherein R¹, R², R^(2′), R³, R⁵, R⁶, and X are as defined above and described herein.

As defined generally above, R² is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂, wherein R and R′ are as defined above and described herein. In some embodiments, R² is hydrogen. In some embodiments, R² is optionally substituted C₁₋₆ aliphatic. In certain embodiments, R² is optionally substituted C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl. In certain embodiments, R² is optionally substituted C₁₋₆ alkyl. In certain embodiments, R² is ethyl. In certain other embodiments, R² is methyl, propyl, isopropyl, butyl, or isobutyl. In some embodiments, R² is C₁₋₆ alkyl substituted with an —OH or —OC₁₋₆ alkyl group. In certain embodiments, R² is —CH₂CH₂OH or —CH₂CH₂OCH₃. In some embodiments, R² is cycloalkyl. In certain embodiments, R² is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, R² is optionally substituted C₁₋₆ alkenyl. In certain embodiments, R² is allyl. In some embodiments, R² is optionally substituted C₁₋₆ alkynyl. In certain embodiments, R² is 2-propynyl. In some embodiments, R² is optionally substituted benzyl. In certain embodiments, R² is unsubstituted benzyl. In certain other embodiments, R² is substituted benzyl. In some embodiments, R² is C₁₋₆ alkyl substituted with an ester group. In certain embodiments, R² is —CH₂CO₂C₁₋₆alkyl or —CH₂CO₂aryl. In certain embodiments, R² is —CH₂CO₂CH₂CH₃. In some embodiments, R² is —OR, —SR, or —N(R′)₂. In certain embodiments, R² is —CN or —NO₂. In some embodiments, R² is halogen. In certain embodiments, R² is fluoro, chloro, bromo, or iodo. In some embodiments, R² is —C(O)R, —CO₂R, —C(O)SR, —C(O)N(R′)₂, —C(O)C(O)R, or —C(O)CH₂C(O)R. In certain embodiments, R² is —C(S)OR or —C(S)N(R′)₂. In other embodiments, R² is —S(O)R, —SO₂R, or —SO₂N(R′)₂. In some embodiments, R² is —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, or —N(R′)C(═N(R′))N(R′)₂. In some embodiments, R² is —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂.

As defined generally above, R^(2′) is —R, —OR, —SR, —N(R′)₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂, wherein R and R′ are as defined above and described herein. In some embodiments, R^(2′) is hydrogen. In some embodiments, R^(2′) is optionally substituted C₁₋₆ aliphatic. In certain embodiments, R^(2′) is optionally substituted C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl. In certain embodiments, R^(2′) is optionally substituted C₁₋₆ alkyl. In certain embodiments, R^(2′) is ethyl. In certain other embodiments, R^(2′) is methyl, propyl, isopropyl, butyl, or isobutyl. In some embodiments, R^(2′) is C₁₋₆ alkyl substituted with an —OH or —OC₁₋₆alkyl group. In certain embodiments, R^(2′) is —CH₂CH₂OH or —CH₂CH₂OCH₃. In some embodiments, R^(2′) is cycloalkyl. In certain embodiments, R^(2′) is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, R^(2′) is optionally substituted C₁₋₆ alkenyl. In certain embodiments, R^(2′) is allyl. In some embodiments, R^(2′) is optionally substituted C₁₋₆ alkynyl. In certain embodiments, R^(2′) is 2-propynyl. In some embodiments, R^(2′) is optionally substituted benzyl. In certain embodiments, R^(2′) is unsubstituted benzyl. In certain other embodiments, R^(2′) is substituted benzyl. In some embodiments, R^(2′) is C₁₋₆ alkyl substituted with an ester group. In certain embodiments, R^(2′) is —CH₂CO₂C₁₋₆alkyl or —CH₂CO₂aryl. In certain embodiments, R^(2′) is —CH₂CO₂CH₂CH₃. In some embodiments, R^(2′) is —OR, —SR, or —N(R′)₂. In some embodiments, R^(2′) is —C(O)R, —CO₂R, —C(O)SR, —C(O)N(R′)₂, —C(O)C(O)R, or —C(O)CH₂C(O)R. In certain embodiments, R^(2′) is —C(S)OR or —C(S)N(R′)₂. In other embodiments, R^(2′) is —S(O)R, —SO₂R, or —SO₂N(R′)₂. In some embodiments, R^(2′) is —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, or —N(R′)C(═N(R′))N(R′)₂. In some embodiments, R^(2′) is —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂.

As defined generally above, R³ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)C(O)R, —C(O)CH₂C(O)R, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —C═NN(R′)₂, —C═NOR, —OC(O)R, or —OC(O)N(R′)₂, wherein R and R′ are as defined above and described herein. In some embodiments, R³ is hydrogen. In some embodiments, R³ is optionally substituted C₁₋₆ aliphatic. In certain embodiments, R³ is optionally substituted C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl. In certain embodiments, R³ is optionally substituted C₁₋₆ alkyl. In certain embodiments, R³ is methyl. In certain other embodiments, R³ is ethyl, propyl, isopropyl, butyl, or isobutyl. In certain embodiments, R³ is —CF₃. In some embodiments, R³ is C₁₋₆ alkyl substituted with an —OH or —OC₁₋₆alkyl group. In certain embodiments, R³ is —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂OCH₂CH₃, —CH₂OCH₃, —CH₂CH₂CH₂OCH₃, —CH(OH)CH₃, or —CH₂CH₂OCH₃. In some embodiments, R³ is C₁₋₆ alkyl substituted with an —NHC₁₋₆alkyl or —N(C₁₋₆alkyl)₂ group. In certain embodiments, R³ is —CH₂NHC₁₋₆ alkyl. In certain embodiments, R³ is —CH₂NHCH₃. In some embodiments, R³ is C₁₋₆ alkyl substituted with an aryl, heteroaryl, carbocyclyl, or heterocyclyl ring. In some embodiments, R³ is optionally substituted benzyl. In certain embodiments, R³ is unsubstituted benzyl. In certain other embodiments, R³ is substituted benzyl. In certain embodiments, R³ is —C(R^(o) ₂Ph. In certain embodiments, R³ is —C(R^(o))₂Ph, wherein R^(o) is hydrogen or methyl. In certain embodiments, R³ is trifluoromethylbenzyl. In certain embodiments, R³ is —C(R^(o))₂(heteroaryl). In certain embodiments, R³ is —C(R^(o))₂(heteroaryl), wherein the heteroaryl is pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, pyridinonyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, thienyl, furanyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, or oxadiazolyl. In certain embodiments, R³ is —CH₂(heteroaryl), wherein the heteroaryl is pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, thienyl, furanyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, or oxadiazolyl. In certain embodiments, R³ is —C(R^(o))₂(carbocyclyl). In certain embodiments, R³ is)-C(R^(o))₂(carbocyclyl), wherein the carbocyclyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl. In certain embodiments, R³ is —CH₂(carbocyclyl), wherein the carbocyclyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl. In certain embodiments, R³ is)-C(R^(o))₂(heterocyclyl). In certain embodiments, R³ is —C(R^(o))₂(heterocyclyl), wherein the heterocyclyl is tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. In certain embodiments, R³ is —CH₂(heterocyclyl), wherein the heterocyclyl is tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. In some embodiments, R³ is optionally substituted C₁₋₆ alkenyl. In certain embodiments, R³ is allyl. In some embodiments, R³ is optionally substituted C₁₋₆ alkynyl. In certain embodiments, R³ is propargyl. In some embodiments, R³ is an optionally substituted aryl or heteroaryl group. In certain embodiments, R³ is phenyl. In certain embodiments, R³ is substituted phenyl. In certain embodiments, R³ is toluoyl. In certain other embodiments, R³ is a 5-6 membered heteroaryl ring having 1-3 heteroatoms selected from nitrogen, oxygen, and sulfur. In certain embodiments, R³ is pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, thienyl, furanyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, or oxadiazolyl. In some embodiments, R³ is —OR, —SR, or —N(R′)₂. In some embodiments, R³ is halogen. In certain embodiments, R³ is fluoro, chloro, bromo, or iodo. In some embodiments, R³ is —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, or —C(O)CH₂C(O)R. In certain embodiments, R³ is optionally substituted —CO₂C₁₋₆alkyl. In certain embodiments, R³ is —CO₂Et or —CO₂Bn. In certain embodiments, R³ is —CONHC₁₋₆alkyl. In certain embodiments, R³ is —CONHCH₃ or —CONHCH₂CH₃. In certain embodiments, R³ is —C(S)OR or —C(S)N(R′)₂. In other embodiments, R³ is —S(O)R, —SO₂R, or —SO₂N(R′)₂. In some embodiments, R³ is —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, or —N(R′)C(═N(R′))N(R′)₂. In some embodiments, R³ is —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂.

In some embodiments, R² and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R² and R³ are taken together with their intervening atoms to form a 5-membered fused ring. In certain embodiments, R² and R³ are taken together with their intervening atoms to form a fused cyclopentene ring. In certain embodiments, R² and R³ are taken together with their intervening atoms to form a 6-membered fused ring. In certain embodiments, R² and R³ are taken together with their intervening atoms to form a fused cyclohexene ring. In certain embodiments, R² and R³ are taken together with their intervening atoms to form a fused benzene ring. In certain embodiments, R² and R³ are taken together with their intervening atoms to form a 5-7 membered partially unsaturated fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R² and R³ are taken together with their intervening atoms to form a 5-7 membered aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R^(2′) and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R^(2′) and R³ are taken together with their intervening atoms to form a 5-membered fused ring. In certain embodiments, R^(2′) and R³ are taken together with their intervening atoms to form a 6-membered fused ring. In certain embodiments, R^(2′) and R³ are taken together with their intervening atoms to form a fused pyridine ring. In certain embodiments, R^(2′) and R³ are taken together with their intervening atoms to form a 5-7 membered partially unsaturated fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R^(2′) and R³ are taken together with their intervening atoms to form a 5-7 membered aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

As defined generally above, X is —N(R⁴)—, —O—, or —S—, wherein R⁴ is as defined above and described herein. In certain embodiments, X is —O— or —S—. In some embodiments, X is —N(R⁴)—. In certain embodiments, X is —NH—. In certain embodiments, X is —N(CH₃)—.

As defined generally above, R⁴ is —R, —C(O)R, —CO₂R, or —S(O)₂R, or R⁴ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered saturated, partially unsaturated, or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R⁴ is hydrogen. In some embodiments, R⁴ is optionally substituted C₁₋₆ alkyl. In certain embodiments, R⁴ is optionally substituted C₁₋₃ alkyl. In certain embodiments, R⁴ is methyl. In certain embodiments, R⁴ is substituted C₁₋₆ alkyl. In certain embodiments, R⁴ is benzyl. In certain embodiments, R⁴ is —CH₂CH₂N(CH₃)₂. In some embodiments, R⁴ is aryl or heteroaryl. In certain embodiments, R⁴ is phenyl. In some embodiments, R⁴ is —C(O)R, —CO₂R, or —S(O)₂R.

In some embodiments, R⁴ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered saturated, partially unsaturated, or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R⁴ and R³ are taken together with their intervening atoms to form a 5-membered fused ring. In certain embodiments, R⁴ and R³ are taken together with their intervening atoms to form a fused pyrrolidine ring. In certain embodiments, R⁴ and R³ are taken together with their intervening atoms to form a 6-membered fused ring. In certain embodiments, R⁴ and R³ are taken together with their intervening atoms to form a fused piperidine ring. In certain embodiments, R⁴ and R³ are taken together with their intervening atoms to form a 5-7 membered partially unsaturated fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R⁴ and R³ are taken together with their intervening atoms to form a 5-7 membered aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

As defined generally above, R⁵ is R, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)C(O)R, or —C(O)CH₂C(O)R, or R⁵ and R² are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R⁵ is hydrogen. In some embodiments, R⁵ is optionally substituted C₁₋₆ alkyl. In certain embodiments, R⁵ is methyl. In certain embodiments, R⁵ is substituted C₁₋₆ alkyl. In certain embodiments, R⁵ is C₁₋₆ alkyl substituted with an —OH or —OC₁₋₆alkyl group. In certain embodiments, R⁵ is —CH₂CH₂OCH₃. In some embodiments, R⁴ is —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)C(O)R, or —C(O)CH₂C(O)R.

As defined generally above, R⁶ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂, wherein R and R′ are as defined above and described herein. In some embodiments, R⁶ is hydrogen. In some embodiments, R⁶ is optionally substituted C₁₋₆ aliphatic. In certain embodiments, R⁶ is optionally substituted C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl. In certain embodiments, R⁶ is optionally substituted C₁₋₆ alkyl. In certain embodiments, R⁶ is ethyl. In certain other embodiments, R⁶ is methyl, propyl, isopropyl, butyl, or isobutyl. In some embodiments, R⁶ is C₁₋₆ alkyl substituted with an —OH or —OC₁₋₆alkyl group. In certain embodiments, R⁶ is —CH₂CH₂OH or —CH₂CH₂OCH₃. In some embodiments, R⁶ is cycloalkyl. In certain embodiments, R⁶ is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, R⁶ is optionally substituted C₁₋₆ alkenyl. In certain embodiments, R⁶ is allyl. In some embodiments, R⁶ is optionally substituted C₁₋₆ alkynyl. In certain embodiments, R⁶ is 2-propynyl. In some embodiments, R⁶ is optionally substituted benzyl. In certain embodiments, R⁶ is unsubstituted benzyl. In certain other embodiments, R⁶ is substituted benzyl. In some embodiments, R⁶ is C₁₋₆ alkyl substituted with an ester group. In certain embodiments, R⁶ is —CH₂CO₂C₁₋₆alkyl or —CH₂CO₂aryl. In certain embodiments, R⁶ is —CH₂CO₂CH₂CH₃. In some embodiments, R⁶ is —OR, —SR, or —N(R′)₂. In certain embodiments, R⁶ is —CN or —NO₂. In some embodiments, R⁶ is halogen. In certain embodiments, R⁶ is fluoro, chloro, bromo, or iodo. In some embodiments, R⁶ is —C(O)R, —CO₂R, —C(O)SR, —C(O)N(R′)₂, —C(O)C(O)R, or —C(O)CH₂C(O)R. In certain embodiments, R⁶ is —C(S)OR or —C(S)N(R′)₂. In other embodiments, R⁶ is —S(O)R, —SO₂R, or —SO₂N(R′)₂. In some embodiments, R⁶ is —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, or —N(R′)C(═N(R′))N(R′)₂. In some embodiments, R⁶ is —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂.

In some embodiments, R⁶ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R⁶ and R³ are taken together with their intervening atoms to form a 5-membered fused ring. In certain embodiments, R⁶ and R³ are taken together with their intervening atoms to form a fused cyclopentene ring. In certain embodiments, R⁶ and R³ are taken together with their intervening atoms to form a 6-membered fused ring. In certain embodiments, R⁶ and R³ are taken together with their intervening atoms to form a fused cyclohexene ring. In certain embodiments, R⁶ and R³ are taken together with their intervening atoms to form a fused benzene ring. In certain embodiments, R⁶ and R³ are taken together with their intervening atoms to form a 5-7 membered partially unsaturated fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R⁶ and R³ are taken together with their intervening atoms to form a 5-7 membered aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

As defined generally above, each R is independently hydrogen or an optionally substituted group selected from C₁₋₆ aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated, partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is hydrogen. In some embodiments, R is optionally substituted C₁₋₆ alkyl, alkenyl, or alkynyl. In certain embodiments, R is optionally substituted C₁₋₆ alkyl. In certain embodiments, R is unsubstituted C₁₋₆ alkyl. In certain embodiments, R is substituted C₁₋₆ alkyl. In certain embodiments, R is methyl, ethyl, propyl, butyl, isopropyl, isobutyl, allyl, or benzyl.

In some embodiments, R is a 3-7 membered saturated or partially unsaturated carbocyclic ring. In certain embodiments, R is a 3-4 membered saturated carbocyclic ring. In other embodiments, R is a 5-7 membered saturated or partially unsaturated carbocyclic ring. In certain embodiments, R is cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, or cycloheptenyl.

In some embodiments, R is a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is a 4-7 membered saturated heterocyclic ring. In other embodiments, R is a 5-7 membered partially unsaturated heterocyclic ring. In certain embodiments, R is tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, or morpholinyl.

In some embodiments, R is an 8-10 membered bicyclic saturated or partially unsaturated carbocylic ring or a 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is decahydronaphthyl, tetrahydronaphthyl, or decalin. In certain other embodiments, R is tetrahydroquinolinyl, tetrahydroisoquinolinyl, or decahydroquinolinyl. In some embodiments, R is a heterocyclyl ring is fused to an aryl or heteroaryl ring. In certain embodiments, R is indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, 2-azabicyclo[2.2.1]heptanyl, octahydroindolyl, or tetrahydroquinolinyl.

In some embodiments, R is phenyl or a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is phenyl. In certain other embodiments, R is a 5-membered heteroaryl ring having 1-3 heteroatoms selected from nitrogen, oxygen, or sulfur. In yet other embodiments, R is a 6-membered heteroaryl ring having 1-3 nitrogens. In certain embodiments, R is phenyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, or triazinyl. In certain other embodiments, R is pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, thienyl, furanyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, or oxadiazolyl.

In some embodiments, R is bicyclic aromatic ring. In certain embodiments, R is naphthyl. In other embodiments, R is an 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is quinolinyl, quinoxalinyl, quinazolinyl, pyridopyrazinyl, or pyridopyrimidyl. In certain other embodiments, R is indolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, benzotriazolyl, benzoxazolyl, benzothiophenyl, indazolyl, imidazopyridyl, imidazopyrimidyl, imidazopyrazinyl, imidazopyridazinyl, pyrazolopyridyl, pyrazolopyrimidyl, pyrazolopyrazinyl, pyrazolopyridazinyl, pyrrolothiazolyl, imidazothiazolyl, thiazolopyridyl, thiazolopyrimidyl, thiazolopypyrazinyl, thiazolopyridazinyl, oxazolopyridyl, oxazolopyrimidyl, oxazolopyrazinyl, or oxazolopyridazinyl.

As defined generally above, each R′ is independently —R, —C(O)R, —CO₂R, or two R′ on the same nitrogen are taken together with the intervening nitrogen to form a 4-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R′ is R as defined and described above. In certain embodiments, R′ is —C(O)R or —CO₂R. In some embodiments, two R′ on the same nitrogen are taken together with their intervening atoms to form a 4-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, two R′ on the same nitrogen are taken together with their intervening atoms to form an azetidine, pyrrolidine, piperidine, morpholine, piperazine, homopiperidine, or homopiperazine ring.

According to one aspect, a provided compound is of formula II:

or a pharmaceutically acceptable salt thereof, wherein R¹, R², R³, and R⁴ are as defined and described herein. In certain embodiments, a compound of formula II has one of the following formulae:

According to another aspect, a provided compound is of formula III:

or a pharmaceutically acceptable salt thereof, wherein R¹, R², R³, and R⁴ are as defined and described herein. In certain embodiments, a compound of formula II has one of the following formulae:

According to another aspect, a provided compound is of formula IV:

or a pharmaceutically acceptable salt thereof, wherein R¹, R², R³, and R⁵ are as defined and described herein. In certain embodiments, R⁵ is optionally substituted C₁₋₆ aliphatic. In certain embodiments, R⁵ is methyl. In some embodiments, R⁵ is optionally substituted C₁₋₆ alkyl. In certain embodiments, R⁵ is substituted C₁₋₆ alkyl. In certain embodiments, R⁵ is C₁₋₆alkyl substituted with —OH or —OC₁₋₆alkyl. In certain embodiments, R⁵ is —CH₂CH₂OMe.

According to another aspect, a provided compound is of formula V:

or a pharmaceutically acceptable salt thereof, wherein R¹, R^(2′), and R³ are as defined and described herein.

Exemplary compounds of formula I are set forth in Table 1 below.

TABLE 1 Exemplary Compounds of Formula I

I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

I-24

I-25

I-26

I-27

I-28

I-29

I-30

I-31

I-32

I-33

I-34

I-35

I-36

I-37

I-38

I-39

I-40

I-41

I-42

I-43

I-44

I-45

I-46

I-47

I-48

I-49

I-50

I-51

I-52

I-53

I-54

I-55

I-56

I-57

I-58

I-59

I-60

I-61

I-62

I-63

I-64

I-65

I-66

I-67

I-68

I-69

I-70

I-71

I-72

I-73

In certain embodiments, the present invention provides any compound depicted in Table 1, above, or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a compound set forth in Table 1-a below.

TABLE 1-a Compounds of Formula I

I-4

I-21

I-23

I-25

I-29

I-30

I-49

In certain embodiments, the present invention provides any compound depicted in Table 1-a, above, or a pharmaceutically acceptable salt thereof.

4. Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

According to another embodiment, the invention provides a composition comprising a provided compound or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle. The amount of compound in compositions of this invention is such that is effective to measurably inhibit a histone demethylase, or a mutant thereof, in a biological sample or in a patient. In some embodiments, the histone demethylase is a 2-oxoglutarate dependent enzyme. In some embodiments, the histone demethylase is a Jumonji domain containing protein. In some embodiments, the histone demethylase is a member of the H3K4 (histone 3K4) demethylase family. In certain embodiments, the histone demethylase is a JARID subfamily of enzymes. In some embodiments, the histone demethylase is selected from JARID1A, JARID1B, or a mutant thereof.

In certain embodiments, the amount of compound in compositions of this invention is such that is effective to measurably inhibit a 2-oxoglutarate dependent enzyme, or a mutant thereof, in a biological sample or in a patient. In certain embodiments, the 2-oxoglutarate dependent enzyme is a Jumonji domain containing protein. In certain embodiments, the Jumonji domain containing protein is a member of the JMJD2 subfamily. In certain embodiments, the member of the JMJD2 subfamily is GASC1.

In certain embodiments, the amount of compound in compositions of this invention is such that is effective to measurably inhibit a member of the H3K4 (histone 3K4) demethylase family of proteins, or a mutant thereof, in a biological sample or in a patient. In certain embodiments, the amount of compound in compositions of this invention is such that is effective to measurably inhibit a member of the JARID subfamily of proteins, or a mutant thereof, in a biological sample or in a patient. In some embodiments, the amount of compound in compositions of this invention is such that is effective to measurably inhibit JARID1A, JARID1B, or a mutant thereof, in a biological sample or in a patient.

In certain embodiments, a composition of this invention is formulated for administration to a patient in need of such composition. In some embodiments, a composition of this invention is formulated for oral administration to a patient.

The term “patient,” as used herein, means an animal, such as a mammal, such as a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle” refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

A “pharmaceutically acceptable derivative” means any non-toxic salt, ester, salt of an ester or other derivative of a compound of this invention that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention or an inhibitorily active metabolite or residue thereof.

As used herein, the term “inhibitorily active metabolite or residue thereof” means that a metabolite or residue thereof is also an inhibitor of a histone demethylase enzyme, or a mutant thereof.

Compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of a compound of the present invention, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

Pharmaceutically acceptable compositions provided by the invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promotors to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

Pharmaceutically acceptable compositions provided by the invention may be formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, pharmaceutically acceptable compositions of this invention are administered without food. In other embodiments, pharmaceutically acceptable compositions of this invention are administered with food.

The amount of provided compounds that may be combined with carrier materials to produce a composition in a single dosage form will vary depending upon the patient to be treated and the particular mode of administration. Provided compositions may be formulate such that a dosage of between 0.01-100 mg/kg body weight/day of the inhibitor can be administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of a provided compound in the composition will also depend upon the particular compound in the composition.

Uses of Compounds and Pharmaceutically Acceptable Compositions

Compounds and compositions described herein are generally useful for the inhibition of activity of one or more enzymes involved in epigenetic regulation.

Epigenetics is the study of heritable changes in gene expression caused by mechanisms other than changes in the underlying DNA sequence. Molecular mechanisms that play a role in epigenetic regulation include DNA methylation and chromatin/histone modifications. Histone methylation, in particular, is critical in many epigenetic phenomena.

Chromatin, the organized assemblage of nuclear DNA and histone proteins, is the basis for a multitude of vital nuclear processes including regulation of transcription, replication, DNA-damage repair and progression through the cell cycle. A number of factors, such as chromatin-modifying enzymes, have been identified that play an important role in maintaining the dynamic equilibrium of chromatin (Margueron, et al. (2005) Curr. Opin. Genet. Dev. 15:163-176).

Histones are the chief protein components of chromatin. They act as spools around which DNA winds, and they play a role in gene regulation. There are a total of six classes of histones (H1, H2A, H2B, H3, H4, and H5) organized into two super classes: core histones (H2A, H2B, H3, and H4) and linker histones (H1 and H5). The basic unit of chromatin is the nucleosome, which consists of about 147 base pairs of DNA wrapped around the histone octamer, consisting of two copies each of the core histones H2A, H2B, H3, and H4 (Luger, et al. (1997) Nature 389:251-260).

Histones, particularly residues of the amino termini of histones H3 and H4 and the amino and carboxyl termini of histones H2A, H2B and H1, are susceptible to a variety of post-translational modifications including acetylation, methylation, phosphorylation, ribosylation sumoylation, ubiquitination, citrullination, deimination, and biotinylation. The core of histones H2A and H3 can also be modified. Histone modifications are integral to diverse biological processes such as gene regulation, DNA repair, and chromosome condensation.

One type of histone modification, demethylation, is catalyzed by histone lysine demethylases (HKDM) or histone arginine demethylases. The Jumonji domain containing family of 2-oxoglutarate dependent oxygenases represents a major class of histone demethylases that are involved in epigenetic regulation. Almost all Jumonji domain containing proteins described to date are histone lysine demethylases, though JMJD6 has been found to be a histone arginine demethylase. An important class of Jumonji domain containing proteins is the JMJD2 (jumonji domain containing 2) subfamily of JMJC-type lysine demethylases.

GASC1 (also known as JMJD2C) is a 2-oxoglutarate dependent histone lysine demethylase in the JMJD2 subfamily. GASC1 demethylates trimethylated lysine 9 and lysine 36 on histone H3 (i.e., H3K9me3 and H3K36me3) (Whetstine, et al. (2006) Cell 125: 467-481). Trimethylation on lysine 9 of histone H3 is associated with heterochromatin formation and transcriptional repression (Cloos, et al. (2006) Nature 442: 307-311). GASC1 is also known to bind to H3K4me3 and H4K20me3 (Huang, et al. (2006) Science 312: 748-751).

In some embodiments, enzymes that are inhibited by the compounds and compositions described herein and against which the methods described herein are useful include 2-oxoglutarate dependent enzymes or an isoform or mutant thereof. In some embodiments, the 2-oxoglutarate dependent enzyme is a Jumonji domain containing protein. In certain embodiments, the Jumonji domain containing protein is a member of the JMJD2 subfamily. In certain embodiments, the member of the JMJD2 subfamily is GASC1.

The activity of a provided compound as an inhibitor of a 2-oxoglutarate dependent enzyme (e.g. Jumonji domain containing protein, e.g. JMJD2, e.g. GASC1) or an isoform or mutant thereof, may be assayed in vitro, in vivo or in a cell line.

In vitro assays include assays that determine inhibition of GASC1 or a mutant thereof. In some embodiments, inhibitor binding may be determined by running a competition experiment where new inhibitors are incubated with GASC1 bound to known radioligands. Detailed conditions for assaying a provided compound as an inhibitor of GASC1 or a mutant thereof are set forth in the Examples below.

In some embodiments, detection of GASC1 activity is achieved with in vitro histone lysine demethylase (HKDM) assays, which can be either direct binding (non-catalytic) or enzymatic (catalytic) assays. Types of substrates that are used in such assays may include: short synthetic peptides corresponding to a number of residues from the N-terminus of histone sequences comprising the target lysine residue, single recombinant histone polypeptides, histone octamers reconstituted with recombinant histone proteins, and reconstituted nucleosomes (using reconstituted octamers and specific recombinant DNA fragments). The reconstituted nucleosomes may be mononucleosomes or oligonucleosomes. 2-Oxoglutarate, a cofactor necessary for GASC1 function, can also be employed in a competitive binding assay. Mass spectrometry and Western blot analysis can also be used to detect GASC1 activity; see for example Whetstine, et al. Cell 125: 467-481 (2006). For examples of HKDM screening assays, see WO 2007/104314 and WO 2008/089883. It will be understood that the assays described herein can be used for other HKDM proteins in addition to GASC1. In certain embodiments, a provided compound is competitive with 2-oxoglutarate.

GASC1 is implicated in proliferative diseases. The GASC1 gene was first identified in esophageal squamous cell carcinoma cell lines, resulting in its designation as gene amplified in squamous cell carcinoma 1 (GASC1) (Yang, et al. (2000) Cancer Res. 60: 4735-4739). Down regulation of GASC1 expression inhibits cell proliferation, and histone methylation regulation is implicated in tumorigenesis (Whetstine, et al. (2006) Cell 125: 467-481). GASC1 interacts with androgen receptor and another histone demethylase, LSD1, in vitro and in vivo and increases androgen receptor-dependent gene expression in prostate cells, implicating GASC1 in prostate cancer (Wissmann, et al. (2007) Nat. Cell Biol. 9: 347-353). Furthermore, the GASC1 gene is amplified in basal like breast tumors and in lung sarcomatoid carcinoma and is translocated in MALT lymphomas (Han, et al. (2008) Genes Chromosomes Cancer 47: 490-499; Helias, et al. (2008) Cancer Genet. Cytogenet. 180: 51-55; Italiano, et al. (2006) Cancer Genet. Cytogenet. 167: 122-130; Vinatzer, et al. (2008) Clin Cancer Res 14: 6426-6431). GASC1 plays an important role in cancer and other proliferative diseases.

As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

In certain embodiments, provided compounds inhibit one or more 2-oxoglutarate dependent enzymes. In certain embodiments, provided compounds inhibit one or more Jumonji domain containing enzymes. In certain embodiments, provided compounds inhibit one or more JMJD2 proteins. In certain embodiments, provided compounds inhibit GASC1. Provided compounds are inhibitors of 2-oxoglutarate dependent enzymes (e.g. GASC1) and are therefore useful for treating one or more disorders associated with activity of a 2-oxoglutarate dependent enzyme (e.g. GASC1). In certain embodiments, the present invention provides a method for treating a GASC1-mediated disorder comprising the step of administering to a patient in need thereof a provided compound, or a pharmaceutically acceptable composition thereof.

As used herein, the term “GASC1-mediated” disorder or condition means any disease or other deleterious condition in which GASC1, or a mutant thereof, is known to play a role. Accordingly, another embodiment of the present invention relates to treating or lessening the severity of one or more diseases in which GASC1, or a mutant thereof, is known to play a role.

Diseases and conditions treatable according to the methods of this invention include, but are not limited to, cancer and other proliferative disorders. In one embodiment, a human patient is treated with a compound of the current invention and a pharmaceutically acceptable carrier, adjuvant, or vehicle, wherein said compound of is present in an amount to measurably inhibit activity of a 2-oxoglutarate dependent enzyme (e.g. Jumonji domain containing protein, e.g. JMJD2, e.g. GASC1).

Another important class of Jumonji domain containing proteins is the H3K4 (histone 3K4) demethylases which are involved in tissue development, cancer, and stem cell biology. (Roesch, et al. (2010) Cell 141:283-594). Such H3K4 demethylases include the JARID subfamily of histone demethylases (e.g., JARID1A and JARID1B).

JARID1A (also known as KDM5A) is highly expressed in the hematopoietic system. JARID1B (also known as KDM5B, PLU-1, and RBP2-H1) is a member of the family of jumonji/ARID1 (JARID1) histone 3K4 demethylases. In normal cells, JARID1B is marginally expressed. However, JARID1B is highly expressed in regenerative tissues such as testis and bone marrow. In cancer, JARID1B functions as a transcriptional regulator of oncogenes, for example BRCA1 in breast cancer (Yamane et al., (2007) Molecular Cell 25:801-812). Indeed, JARID is overexpressed in breast cancer. It was also reported that JARID1B is highly expressed in slow-cycling melanoma cells. Accordingly, inhibition of JARID1B is an important target for eradicating all melanoma cells (rapidly proliferating and slow-cycling) (Roesch, et al. (2010) Cell 141:283-594).

In some embodiments, enzymes that are inhibited by the compounds and compositions described herein and against which the methods described herein are useful include 2-oxoglutarate dependent enzymes or an isoform or mutant thereof. In some embodiments, the 2-oxoglutarate dependent enzyme is a Jumonji domain containing protein. In certain embodiments, the Jumonji domain containing protein is a member of the JMJD2 subfamily. In certain embodiments, the member of the JMJD2 subfamily is GASC1. In some embodiments, the enzyme is a member of the JARID subfamily. In certain embodiments, the enzyme is JARID1A, PLU-1, or JMJD2B.

The activity of a provided compound as an inhibitor of a histone demethylase enzyme (e.g. Jumonji domain containing protein, e.g. JMJD2, JMJD2B, JARID1A, JARID1B, PLU-1, or GASC1) or an isoform or mutant thereof, may be assayed in vitro, in vivo or in a cell line.

In vitro assays include assays that determine inhibition of an enzyme or a mutant thereof. In some embodiments, inhibitor binding may be determined by running a competition experiment where new inhibitors are incubated with the enzyme bound to known radioligands. Detailed conditions for assaying a provided compound as an inhibitor of enzyme or a mutant thereof are set forth in the Examples below.

In some embodiments, detection of histone demethylase (e.g. Jumonji domain containing protein, e.g. JMJD2, JMJD2B, JARID1A, JARID1B, PLU-1, or GASC1) activity is achieved with in vitro histone lysine demethylase (HKDM) assays, which can be either direct binding (non-catalytic) or enzymatic (catalytic) assays. Types of substrates that are used in such assays may include: short synthetic peptides corresponding to a number of residues from the N-terminus of histone sequences comprising the target lysine residue, single recombinant histone polypeptides, histone octamers reconstituted with recombinant histone proteins, and reconstituted nucleosomes (using reconstituted octamers and specific recombinant DNA fragments). The reconstituted nucleosomes may be mononucleosomes or oligonucleosomes. 2-Oxoglutarate, a cofactor necessary for GASC1 function, can also be employed in a competitive binding assay. Mass spectrometry and Western blot analysis can also be used to detect GASC1 activity; see for example Whetstine, et al. Cell 125: 467-481 (2006). For examples of HKDM screening assays, see WO 2007/104314 and WO 2008/089883. It will be understood that the assays described herein can be used for other HKDM proteins in addition to GASC1. In certain embodiments, a provided compound is competitive with 2-oxoglutarate.

In certain embodiments, provided compounds inhibit one or more 2-oxoglutarate dependent enzymes. In certain embodiments, provided compounds inhibit one or more Jumonji domain containing enzymes. In certain embodiments, provided compounds inhibit one or more JMJD2 proteins. In certain embodiments, provided compounds inhibit GASC1. In some embodiments, provided compounds inhibit one or more of JARID1A, JARID1B, PLU-1, and/or JMJD2B. Provided compounds are inhibitors of such histone demethylases and are therefore useful for treating one or more disorders associated with activity of one or more of JARID1A, JARID1B, PLU-1, and/or JMJD2B. In certain embodiments, the present invention provides a method for treating a JARID1A-, JARID1B-, PLU-1-, and/or JMJD2B-mediated disorder comprising the step of administering to a patient in need thereof a provided compound, or a pharmaceutically acceptable composition thereof.

As used herein, the term “JARID1A-mediated” disorder or condition means any disease or other deleterious condition in which JARID1A, or a mutant thereof, is known to play a role. Accordingly, another embodiment of the present invention relates to treating or lessening the severity of one or more diseases in which JARID1A, or a mutant thereof, is known to play a role.

As used herein, the term “JARID1B-mediated” disorder or condition means any disease or other deleterious condition in which JARID1B, or a mutant thereof, is known to play a role. Accordingly, another embodiment of the present invention relates to treating or lessening the severity of one or more diseases in which JARID1B, or a mutant thereof, is known to play a role.

As used herein, the term “PLU-1-mediated” disorder or condition means any disease or other deleterious condition in which PLU-1, or a mutant thereof, is known to play a role. Accordingly, another embodiment of the present invention relates to treating or lessening the severity of one or more diseases in which PLU-1, or a mutant thereof, is known to play a role.

As used herein, the term “JMJD2B-mediated” disorder or condition means any disease or other deleterious condition in which JMJD2B, or a mutant thereof, is known to play a role. Accordingly, another embodiment of the present invention relates to treating or lessening the severity of one or more diseases in which JMJD2B, or a mutant thereof, is known to play a role.

The invention further relates to a method for treating, ameliorating or preventing cancer or another proliferative disorder by administration of an effective amount of a compound according to this invention to a mammal, in particular a human in need of such treatment. In some aspects of the invention, the disease to be treated by the methods of the present invention may be cancer. Examples of cancers that may be treated using the compounds and methods described herein include, but are not limited to, adrenal cancer, acinic cell carcinoma, acoustic neuroma, acral lentigious melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, androgen dependent cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear-cell sarcoma of the kidney, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine gland neoplasm, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hemangioblastoma, head and neck cancer, hemangiopericytoma, hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, leukemia, leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myelogeous leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non-small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor, mucinous tumor, multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, ocular cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma, pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, preimary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma periotonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease, small intestine cancer, stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroid cancer, transitional cell carcinoma, throat cancer, urachal cancer, urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor.

In some embodiments, the present invention provides a method for the treatment of benign proliferative disorder. Examples of benign proliferative disorders treated with compounds according to the present invention include, but are not limited to, benign soft tissue tumors, bone tumors, brain and spinal tumors, eyelid and orbital tumors, granuloma, lipoma, meningioma, multiple endocrine neoplasia, nasal polyps, pituitary tumors, prolactinoma, pseudotumor cerebri, seborrheic keratoses, stomach polyps, thyroid nodules, cystic neoplasms of the pancreas, hemangiomas, vocal cord nodules, polyps, and cysts, Castleman disease, chronic pilonidal disease, dermatofibroma, pilar cyst, pyogenic granuloma, and juvenile polyposis syndrome.

The invention further provides a method for the treatment a subject, such as a human, suffering from one of the abovementioned conditions, illnesses, disorders or diseases. The method comprises administering a therapeutically effective amount of one or more of the compounds according to this invention, which function by inhibiting one or more 2-oxoglutarate dependent enzymes (e.g. Jumonji domain containing protein, e.g. JMJD2, e.g. GASC1) and, in general, by modulating protein methylation, to induce various cellular effects, in particular induction or repression of gene expression, arresting cell proliferation, inducing cell differentiation and/or inducing apoptosis, to a subject in need of such treatment.

The invention further provides a therapeutic method useful for modulating protein methylation, gene expression, cell proliferation, cell differentiation and/or apoptosis in vivo in diseases mentioned above, in particular cancer, comprising administering to a subject in need of such therapy a pharmacologically active and therapeutically effective amount of one or more of the compounds according to this invention.

The invention further provides a method for regulating endogenous or heterologous promotor activity by contacting a cell with a compound according to this invention.

The invention further relates to the use of provided compounds for the production of pharmaceutical compositions which are employed for the treatment and/or prophylaxis and/or amelioration of the diseases, disorders, illnesses and/or conditions as mentioned herein.

The invention further relates to the use of provided compounds for the production of pharmaceutical compositions which are employed for the treatment and/or prophylaxis of diseases and/or disorders responsive or sensitive to the inhibition of histone demethylases, particularly those diseases mentioned above, such as e.g. cancer.

Provided compounds or compositions may be administered using any amount and any route of administration effective for treating or lessening the severity of cancer or other proliferative disorder. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular agent, its mode of administration, and the like. Compounds of the invention are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. The expression “unit dosage form” as used herein refers to a physically discrete unit of agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts.

Pharmaceutically acceptable compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, as an oral or nasal spray, or the like, depending on the severity of the infection being treated. In certain embodiments, the compounds of the invention may be administered orally or parenterally at dosage levels of about 0.01 mg/kg to about 50 mg/kg and, in certain embodiments, from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

According to one embodiment, the invention relates to a method of inhibiting one or more histone demethylase (e.g. Jumonji domain containing protein, e.g. JMJD2, JMJD2B, JARID1A, JARID1B, PLU-1e.g. GASC1) activity in a biological sample comprising the step of contacting said biological sample with a provided compound, or a composition comprising said compound.

According to another embodiment, the invention relates to a method of inhibiting a histone demethylase (e.g. Jumonji domain containing protein, e.g. JMJD2, JMJD2B, JARID1A, JARID1B, PLU-1e.g. GASC1), or a mutant thereof, activity in a biological sample comprising the step of contacting said biological sample with a provided compound, or a composition comprising said compound.

The term “biological sample”, as used herein, includes, without limitation, cell cultures or extracts thereof; biopsied material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

Inhibition of activity of a histone demethylase (e.g. Jumonji domain containing protein, e.g. JMJD2, JMJD2B, JARID1A, JARID1B, PLU-1e.g. GASC1) or a mutant thereof, in a biological sample is useful for a variety of purposes that are known to one of skill in the art. Examples of such purposes include, but are not limited to, blood transfusion, organ-transplantation, biological specimen storage, and biological assays.

According to another embodiment, the invention relates to a method of inhibiting activity of a histone demethylase (e.g. Jumonji domain containing protein, e.g. JMJD2, JMJD2B, JARID1A, JARID1B, PLU-1e.g. GASC1), or a mutant thereof, in a patient comprising the step of administering to said patient a provided compound, or a composition comprising said compound. In certain embodiments, the present invention provides a method for treating a disorder mediated by a histone demethylase (e.g. Jumonji domain containing protein, e.g. JMJD2, JMJD2B, JARID1A, JARID1B, PLU-1e.g. GASC1), or a mutant thereof, in a patient in need thereof, comprising the step of administering to said patient a compound according to the present invention or pharmaceutically acceptable composition thereof. Such disorders are described in detail herein.

Depending upon the particular condition, or disease, to be treated, additional therapeutic agents that are normally administered to treat that condition may also be present in the compositions of this invention or administered separately as a part of a dosage regimen. As used herein, additional therapeutic agents that are normally administered to treat a particular disease, or condition, are known as “appropriate for the disease, or condition, being treated.”

In some embodiments, the additional therapeutic agent is an epigenetic drug. As used herein, the term “epigenetic drug” refers to a therapeutic agent that targets an epigenetic regulator. Example of epigenetic regulators include the histone demethylase (e.g. Jumonji domain containing protein, e.g. JMJD2, JMJD2B, JARID1A, JARID1B, PLU-1e.g. GASC1) already described, as well as other histone demethylases, histone lysine methyl transferases, histone arginine methyl transferases, histone deacetylases, histone acetylases, histone methylases, and DNA methyltransferases. Histone deacetylase inhibitors include, but are not limited to, vorinostat.

For example, compounds of the present invention, or a pharmaceutically acceptable composition thereof, are administered in combination with chemotherapeutic agents to treat proliferative diseases and cancer. Examples of known chemotherapeutic agents include, but are not limited to, doxorubicin, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, interferons, platinum derivatives, taxanes (e.g., paclitaxel, docetaxel), vinca alkaloids (e.g., vinblastine), anthracyclines (e.g., doxorubicin), epipodophyllotoxins (e.g., etoposide), cisplatin, an mTOR inhibitor (e.g., a rapamycin), methotrexate, actinomycin D, dolastatin 10, colchicine, trimetrexate, metoprine, cyclosporine, daunorubicin, teniposide, amphotericin, alkylating agents (e.g., chlorambucil), 5-fluorouracil, camptothecin, cisplatin, metronidazole, and imatinib mesylate, among others. In other embodiments, a compound of the present invention is administered in combination with a biologic agent, such as bevacizumab or panitumumab.

In certain embodiments, compounds of the present invention, or a pharmaceutically acceptable composition thereof, are administered in combination with an antiproliferative or chemotherapeutic agent selected from any one or more of abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, BCG live, bevacuzimab, fluorouracil, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, camptothecin, carboplatin, carmustine, cetuximab, chlorambucil, cladribine, clofarabine, cyclophosphamide, cytarabine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin, dexrazoxane, docetaxel, doxorubicin (neutral), doxorubicin hydrochloride, dromostanolone propionate, epirubicin, epoetin alfa, elotinib, estramustine, etoposide phosphate, etoposide, exemestane, filgrastim, floxuridine, fludarabine, fulvestrant, gefitinib, gemcitabine, gemtuzumab, goserelin acetate, histrelin acetate, hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib mesylate, interferon alfa-2a, interferon alfa-2b, irinotecan, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, megestrol acetate, melphalan, mercaptopurine, 6-MP, mesna, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nelarabine, nofetumomab, oprelvekin, oxaliplatin, paclitaxel, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin, porfimer sodium, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, sorafenib, streptozocin, sunitinib maleate, talc, tamoxifen, temozolomide, teniposide, VM-26, testolactone, thioguanine, 6-TG, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, ATRA, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, zoledronate, or zoledronic acid.

Other examples of agents the inhibitors of this invention may also be combined with include, without limitation: treatments for Alzheimer's Disease such as donepezil hydrochloride and rivastigmine; treatments for Parkinson's Disease such as L-DOPA/carbidopa, entacapone, ropinrole, pramipexole, bromocriptine, pergolide, trihexephendyl, and amantadine; agents for treating multiple sclerosis (MS) such as beta interferon (e.g., Avonex® and Rebie), glatiramer acetate, and mitoxantrone; treatments for asthma such as albuterol and montelukast sodium; agents for treating schizophrenia such as zyprexa, risperdal, seroquel, and haloperidol; anti-inflammatory agents such as corticosteroids, TNF blockers, IL-1 RA, azathioprine, cyclophosphamide, and sulfasalazine; immunomodulatory and immunosuppressive agents such as cyclosporin, tacrolimus, rapamycin, mycophenolate mofetil, interferons, corticosteroids, cyclophophamide, azathioprine, and sulfasalazine; neurotrophic factors such as acetylcholinesterase inhibitors, MAO inhibitors, interferons, anti-convulsants, ion channel blockers, riluzole, and anti-Parkinsonian agents; agents for treating cardiovascular disease such as beta-blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers, and statins; agents for treating liver disease such as corticosteroids, cholestyramine, interferons, and anti-viral agents; agents for treating blood disorders such as corticosteroids, anti-leukemic agents, and growth factors; and agents for treating immunodeficiency disorders such as gamma globulin.

In certain embodiments, compounds of the present invention, or a pharmaceutically acceptable composition thereof, are administered in combination with a monoclonal antibody or an siRNA therapeutic.

Those additional agents may be administered separately from an inventive compound-containing composition, as part of a multiple dosage regimen. Alternatively, those agents may be part of a single dosage form, mixed together with a compound of this invention in a single composition. If administered as part of a multiple dosage regime, the two active agents may be submitted simultaneously, sequentially or within a period of time from one another normally within five hours from one another.

As used herein, the term “combination,” “combined,” and related terms refers to the simultaneous or sequential administration of therapeutic agents in accordance with this invention. For example, a compound of the present invention may be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present invention provides a single unit dosage form comprising a compound of formula I, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

The amount of both an inventive compound and additional therapeutic agent (in those compositions which comprise an additional therapeutic agent as described above) that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. In certain embodiments, compositions of this invention are formulated such that a dosage of between 0.01-100 mg/kg body weight/day of an inventive can be administered.

In those compositions which comprise an additional therapeutic agent, that additional therapeutic agent and the compound of this invention may act synergistically. Therefore, the amount of additional therapeutic agent in such compositions will be less than that required in a monotherapy utilizing only that therapeutic agent. In certain embodiments, in such compositions a dosage of between 0.01-1,000 μg/kg body weight/day of the additional therapeutic agent can be administered.

The amount of additional therapeutic agent present in the compositions of this invention will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. In certain embodiments, the amount of additional therapeutic agent in the presently disclosed compositions will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent.

The compounds of this invention, or pharmaceutical compositions thereof, may also be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents and catheters. Vascular stents, for example, have been used to overcome restenosis (re-narrowing of the vessel wall after injury). However, patients using stents or other implantable devices risk clot formation or platelet activation. These unwanted effects may be prevented or mitigated by pre-coating the device with a pharmaceutically acceptable composition comprising an inhibitor of a histone demethylase (e.g. Jumonji domain containing protein, e.g. JMJD2, JMJD2B, JARID1A, JARID1B, PLU-1e.g. GASC1). Implantable devices coated with a compound of this invention are another embodiment of the present invention.

EXEMPLIFICATION

As depicted in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures. It will be appreciated that, although the general methods depict the synthesis of certain compounds of the present invention, the following general methods, and other methods known to one of ordinary skill in the art, can be applied to all compounds and subclasses and species of each of these compounds, as described herein.

Example 1 Synthesis of ethyl 2-acetylpentanoate

Ethyl 2-acetylpentanoate was synthesized according to Beddow et al, Org. Biomol. Chem. 5: 2812-2825 (2007). To a solution of t-BuOK (11.8 g, 0.11 mol) in THF (150 mL) was added ethyl 3-oxobutanoate (13 g, 0.1 mol) dropwise at 0° C., after stirred for 30 minutes, 1-bromopropane (12.3 g, 0.1 mmol) was added dropwise and the mixture was refluxed for 16 h. The reaction mixture was quenched by water, extracted with EtOAc (100 mL×2), combined organic layer was dried over anhydrous Na₂SO₄, evaporated, purified by column chromatography to give the expected compound ethyl 2-acetylpentaoate (8.5 g, 49%) as colorless oil. m/z (ESI) 173 [M+H]⁺.

Example 2

By a similar method to Example 1, using the appropriate starting materials, the compounds in Table 2 were prepared.

TABLE 2 Compound name Structure Data ethyl 2-acetyl-4- methylpentanoate

m/z (ESI) 187 [M + H]⁺ ethyl 2- acetylpent-4- enoate

m/z (ESI) 171 [M + H]⁺ ethyl 2-acetyl-4- methoxybutanoate

m/z (ESI) 189 [M + H]⁺ ethyl 2- acetylpent-4- ynoate

m/z (ESI) 169 [M + H]⁺ ethyl 2-ethyl- 4,4,4-trifluoro-3- oxobutanoate

m/z (ESI) 213 [M + H]⁺ ethyl 4-ethoxy-2- ethyl-3- oxobutanoate

m/z (ESI) 203 [M + H]⁺ ethyl 2-(2- methoxyacetyl) pent-4-enoate

m/z (ESI) 201 [M + H]⁺ ethyl 2- benzoylbutanoate

m/z (ESI) 221 [M + H]⁺ ethyl 2-(furan-2- carbonyl) butanoate

m/z (ESI) 211 [M + H]⁺ 3-ethylpentane- 2,4-dione

m/z (ESI) 129 [M + H]⁺

Example 3 Synthesis of ethyl 2-ethyl-4-methoxy-3-oxobutanoate

Ethyl 2-ethyl-4-methoxy-3-oxobutanoate was prepared according to WO 98/43968. Zinc (2 g, 30 mmol), methoxyacetonitrile (1.42 g, 20 mmol) and a catalytic amount of mercuric chloride in toluene (50 mL) were heated to reflux. Ethyl 2-bromobutanoate (5.85 g, 30 mmol) was added dropwise, then reflux continued for a hour, and cooled to a room temperature. 10% Aqueous sulfuric solution (16.5 mL) was added, and the organic layer was separated. The aqueous layer was further extracted with ethyl ether and the combined organic layers washed with water and saturated sodium bicarbonate solution, then dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography to give the product as yellow oil (1.7 g, 45%). ¹H NMR (300 MHz, CDCl₃) δ 4.14 (q, J=7.2 Hz, 2H), 4.07 (d, J=3.6 Hz, 2H), 3.45 (t, J=7.2 Hz, 1H), 3.37 (s, MI), 1.85 (m, 2H), 1.22 (t, J=7.2 Hz, 3H). 0.90 (t, J=7.5 Hz, 3H); m/z (ESI) 189 [M+H]⁺.

Example 4

By a similar method to Example 3, using the appropriate starting materials, the compounds in Table 3 were prepared and isolated.

TABLE 3 Compound name Structure Data ethyl 2-(2- methoxyacetyl) pentanoate

m/z (ESI) 225 [M + Na⁺] ethyl 2-ethyl-3- oxo-4- phenylbutanoate

m/z (ESI) 257 [M + Na⁺]

Example 5 Synthesis of diethyl 2-ethyl-3-oxosuccinate

Diethyl-2-ethyl-3-oxosuccinate was prepared according to Soloway et al, J. Org. Chem. 69: 2677-2678 (1947). To a mixture of NaH (60%, 12 g, 300 mmol) and diethyl oxalate (43.8 g, 300 mmol) in ether (100 mL), ethyl butyrate (18 g, 150 mmol) was added. The reaction mixture was refluxed over night. After cooling to room temperature water was added, the mixture was extracted with EtOAc. The organic layer was dried over with Na₂SO₄ and evaporated in vacuo. The residue was purified by column chromatography to give diethyl-2-ethyl-3-oxosuccinate (8 g, 24%) as light oil. m/z (ESI) 217 [M+H]⁺.

Example 6 Synthesis of ethyl 2-ethyl-6-methoxy-3-oxohexanoate

Ethyl 2-ethyl-6-methoxy-3-oxohexanoate was prepared according to WO2006124490.

Synthesis of ethyl 6-methoxy-3-oxohexanoate

To a solution of ethyl 3-oxobutanoate (1.3 g, 10 mol) in THF (50 mL) was added NaH (60%, 480 mg, 12 mmol) at 0° C. After stirring under N₂ at 0° C. for 0.5 h, n-BuLi (4 mL, 10 mmol) was added at 0° C. and then the solution of the mixture was cooled to −25° C. After 1-bromo-2-methoxyethane (1.39 g, 10 mmol) was added, the solution of the mixture was stirring for overnight at room temperature. The mixture was evaporated in vacuo, purified by column chromatography to give ethyl 6-methoxy-3-oxohexanoate (0.65 g, 34.5%). m/z (ESI) 211 [M+Na]⁺.

Synthesis of ethyl 2-ethyl-6-methoxy-3-oxohexanoate

To a solution of ethyl 6-methoxy-3-oxohexanoate (650 mg, 3.45 mmol) in THF (50 mL), ^(t)BuOK (406 mg, 3.63 mmol) was added at 0° C. and then the solution of the mixture was stirring for 30 min at 0° C., followed by refluxing overnight. The mixture was evaporated in vacuo, purified by column chromatography to give ethyl 2-ethyl-6-methoxy-3-oxohexanoate (400 mg, 53.6%). m/z (ESI) 217 [M+H]⁺.

Example 7 Synthesis of 6-cyclopropyl-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile

Synthesis of 2-cyclopropyl-3-oxobutanenitrile

To a solution of 2-cyclopropylacetonitrile (1.17 g, 14.4 mmol) in THF (10 mL) was added LDA (8.7 ml, 17.3 mmol) dropwise at −78° C. under N₂. After stirred for 60 mins, (CH₃O)₂O (1.12 g, 14.4 mmol) was added dropwise at −78° C. and the mixture was stirred for 2 h at ambient temperature. The reaction mixture was quenched by the aqueous of HCl (2N), extracted with ethyl acetate (30 ml×3), combined organic layer was dried over anhydrous Na₂SO₄ and evaporated, purified by column chromatography to give 2-cyclopropyl-3-oxobutanenitrile as yellow oil. m/z (ESI) 124 [M+H]⁺.

Synthesis of ethyl 2-cyclopropyl-3-oxobutanoate

To a solution of 2-cyclopropyl-3-oxobutanenitrile (600 mg, 5 mmol) in EtOH (10 mL) was added acetyl chloride (3 mL) dropwise at 0° C. After stirred for 16 h, EtOH was removed, the mixture was added concentrated HCl (1 mL) and EtOH (10 mL), and stirred for 4 h at 40° C. The mixture was quenched by water and extracted with ethyl acetate (20 mL×3), combined organic layer was dried over anhydrous Na₂SO₄ and evaporated, purified by column chromatography to give ethyl 2-cyclopropyl-3-oxobutanoate (30 mg, 10%) as yellow oil. m/z (ESI) 171 [M+H]⁺.

Example 8 Synthesis of 5-amino-3-ethyl-1H-pyrazole-4-carbonitrile

5-Amino-3-ethyl-1H-pyrazole-4-carbonitrile was prepared in a manner substantially similar to that described in WO2005070916 and US2006135526.

Synthesis of 2-(1-methoxypropylidene)malononitrile

A mixture of malononitrile (180 g, 1.02 mol,) and triethyl orthopropionate (66 g, 1 mol) was refluxed for 3 h. The reaction mixture was distilled under vacuum to give the expected compound 12-1-a (60 g, 40%) as pale yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 4.46 (q, J=6.9 Hz, 2H), 2.65 (q, J=7.5 Hz, 2H), 1.45 (t, J=6.9 Hz, 3H), 1.26 (t, J=7.5 Hz, 3H).

Synthesis of 5-amino-3-ethyl-1H-pyrazole-4-carbonitrile

A solution of 2-(1-methoxypropylidene)malononitrile (10 g, 0.067 mol) in EtOH (50 mL) was added dropwise into the solution of hydrazine monohydrate (6.8 ml, 0.134 mol) in EtOH (100 mL) at 0° C. for 30 min After stirred for 3 h at 90° C., the mixture was concentrated and purified by column chromatography to give the expected compound 5-amino-3-ethyl-1H-pyrazole-4-carbonitrile (5 g, 60% yield) as yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 4.40 (s, 2H), 2.69 (q, J=7.5 Hz, 2H), 1.29 (t, J=7.5 Hz, 3H).

Example 9

General Procedure

A mixture of a cyanopyrazole (0.86 mmol), a beta-ketoester (293 mg, 1.72 mmol) and acetic acid (3 mL) is stirred at 80° C. for 1.5 hours. The mixture is cooled to room temperature. The solvent is removed in vacuo. The residue is purified by silica gel column chromatography to yield the desired compound.

Using the general procedure above and the appropriate starting materials, the compounds in Table 4 were prepared.

TABLE 4 Compound Name Structure Data 2-ethyl-9-oxo- 4,5,6,7,8,9- hexahydropyrazolo[5,1- b]quinazoline-3- carbonitrile

¹H NMR (300 MHz, DMSO) δ 12.84 (brs, 1H), 2.74 (q, J = 6.9 Hz, 2H,), 2.62 (m, 2H), 2.40 (m, 2H) 1.71 (m, 4H), 1.26 (t, J = 6.9 Hz, 3H); m/z (ESI) 243 [M + H]⁺. 2-methyl-9-oxo- 4,5,6,7,8,9- hexahydropyrazolo[5,1- b]quinazoline-3- carbonitrile

¹H NMR (300 MHz, CD₃OD) δ 2.72 (m, 2H), 2.60 (m, 2H), 2.43 (s, 3H), 1.81 (m, 4H). m/z (ESI) 229 [M + H]⁺. 9-oxo-4,5,6,7,8,9- hexahydropyrazolo[5,1- b]quinazoline-3- carbonitrile

¹H NMR (300 MHz, DMSO-d6) δ 13.07 (s, 1H), 8.32 (s, 1H), 2.66 (m, 2H), 2.42 (m, 2H), 1.72 (m, 4H). 6-isopropyl-2,5- dimethyl-7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H-NMR (300 MHz, CD₃COCD₃) δ 3.11 (m, 1H), 2.49 (s, 3H), 2.36 ( s, 1H) 1.34 (d, J = 6.9 Hz, 6H); m/z (ESI) 231 [M + H]⁺. 6-propyl-2,5-dimethyl- 7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, CD₃COCD₃) δ 2.54 (t, J = 7.5 Hz, 2H), 2.49 (s, 3H), 2.36 (s, 3H), 2.06 (m, 2H), 1.55 (m, 2H), 0.96 (t, J = 7.5 Hz, 3H); m/z (ESI) 231 [M + H]⁺. 6-isopropyl-5-methyl- 7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, DMSO-d6) δ 7.93 (s, 1H), 3.02 (m, 1H), 2.26 (s, 3H), 1.27 (d, J = 7.2 Hz, 6H); m/z (ESI) 217 [M + H]⁺. 6-propyl-5-methyl-7- oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, DMSO-d6) δ 13.07 (s, 1H), 8.35 (s, 1H), 2.47 (m, 2H), 2.38 (s, 3H), 1.46 (q, J = 7.5 Hz, 2H), 0.92 (t, J = 7.5 Hz, 3H); m/z (ESI) 217 [M + H]⁺. 6-ethyl-5- (methoxymethyl)-2- methyl-7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, DMSO) δ 4.26 (s, 2H), 3.27 (s, 3H), 2.48 (m, 2H), 2.29 (s, 3H), 1.01 (t, J = 7.2 Hz, 3H); m/z (ESI) 247 [M + H]⁺. 6-ethyl-5- (methoxymethyl)-7- oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, CD₃COCD₃) δ 7.93 (s, 1H), 4.40 (s, 2H), 3.34 (s, 3H), 2.65 (q, J = 7.5 Hz, 2H), 1.12 (t, J = 7.5 Hz, 3H); m/z (ESI) 233 [M + H]⁺. ethyl 3-cyano-6-ethyl- 2-methyl-7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-5- carboxylate

¹H NMR (300 MHz, CD₃OD) δ 4.52 (q, J = 7.2 Hz, 2H), 2.82 (q, J = 7.2 Hz, 2H), 2.50 (s, 3H), 1.47 (t, J = 7.2 Hz, 3H), 1.21 (t, J = 7.5 Hz, 3H); m/z (ESI) 275 [M + H]⁺. ethyl 3-cyano-6-ethyl- 7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-5- carboxylate

¹H NMR (300 MHz, CD₃OD) δ 8.26 (s, 1H), 4.44 (q, J = 7.2 Hz, 2H), 2.70 (q, J = 6.9 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H), 1.17 (t, J = 7.2 Hz, 3H); m/z (ESI) 262 [M + H]⁺. 6-ethyl-5-methyl-7- oxo-4,7-dihydro- [1,2,3]triazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, CD₃OD) δ 2.82 (s, 3H), 2.69 (q, J = 7.5 Hz, 2H), 1.20 (t, J = 7.5 Hz, 3H); m/z (ESI) 204 [M + H]⁺. 5-methyl-7-oxo-6- propyl-4,7-dihydro- [1,2,3]triazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, CD₃OD) δ 2.82 (s, 3H), 2.65 (t, J = 7.5 Hz, 3H), 1.63 (m, 2H), 1.06 (t, J = 7.2 Hz, 3H); m/z (ESI) 218 [M + H]⁺. 5-(methoxymethyl)-7- oxo-6-propyl-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, CD₃OD) δ 8.29 (s, 1H), 4.78 (s, 2H), 3.59 (s, 3H), 2.63 (m, 2H), 1.64 (m, 2I-I), 1.05 (t, J = 7.5 Hz, 3H); m/z (ESI) 247 [M + H]⁺. 5-(ethoxymethyl)-6- ethyl-7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹HNMR (300 MHz, CD₃OD) δ 8.27 (s, 1H), 4.64 (s, 2H), 3.71 (q, J = 7.2 Hz, 2H), 2.69 (q, J = 7.5 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H), 1.20 (t, J = 7.5 Hz, 3H); m/z (ESI) 247 [M + H]⁺. 6-(2-methoxyethyl)-5- methyl-7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, CD₃OD) δ 8.26 (s, 1H), 3.62 (t, J = 6.6 Hz, 2H), 3.39 (s, 3H), 2.93 (t, J = 6.6 Hz, 2H), 2.55 (s, 3H); m/z (ESI) 233 [M + H]⁺. 6-ethyl-5-(3- methoxypropyl)-7-oxo- 4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, CD₃Cl) δ 11.87 (brs, 1H), 8.01 (s, 1H), 3.65 (m, 2H), 3.63 (s, 3H), 2.96 (m, 2H), 2.66 (q, J = 7.5 Hz, 2H), 2.08 (m, 2H), 1.13 (t, J = 7.5 Hz, 3H); m/z (ESI) 261 [M + H]⁺. 5-methyl-7-oxo-6- (prop-2-ynyl)-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹HNMR (300 MHz, CD₃OD) δ 8.26 (s, 1H), 3.58 (d, J = 2.7 Hz, 2H), 2.59 (s, 3H), 2.37 (t, J = 2.7 Hz, 1H); m/z (ESI) 235 [M + Na]⁺. 6-allyl-5- (methoxymethyl)-7- oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹HNMR (300 MHz, CD₃Cl) δ 9.38 (brs, 1H), 8.07 (s, 1H), 5.83 (m, 1H), 5.08 (m, 2H), 4.55 (s, 2H), 3.63 (s, 3H), 3.29 (d, J = 6.3 Hz, 2H); m/z (ESI) 267 [M + Na]+. 6-ethyl-7-oxo-5- phenyl-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹HNMR (300 MHz, CD₃OD) δ 8.30 (s, 1H), 7.64-7.57 (5H), 2.49 (q, J = 7.2 Hz, 2H), 1.14 (t, J = 7.2 Hz, 3H); m/z (ESI) 265 [M + H]⁺. 5-benzyl-6-ethyl-7-oxo- 4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹HNMR (300 MHz, CD₃OD) δ 8.26 (s, 1H), 7.39-7.27 (5H), 4.20 (s, 2H), 2.65 (q, J = 7.5 Hz, 2H), 1.03 (t, J = 7.5 Hz, 3H); m/z (ESI) 279 [M + H]⁺. benzyl 3-cyano-6-ethyl- 7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-5- carboxylate

¹HNMR (300 MHz, CD₃OD) δ 8.22 (s, 1H), 7.56 ? 7.42 (5H), 5.51 (s, 2H), 2.79 (q, J = 7.2 Hz, 2H), 1.12 (t,J = 7.2 Hz, 3H); m/z (ESI) 245 [M + H]⁺. ethyl 3-cyano-7-oxo-6- propyl-4,7- dihydropyrazolo[1,5- a]pyrimidine-5- carboxylate

¹HNMR (300 MI-lz, CD₃OD) δ 8.17 (s, 1H), 4.43 (q, J = 7.2 Hz, 2H), 2.62 (t, J = 7.5 Hz, 2H), 1.62 (m, 2H), 1.44 (t, J = 7.2 Hz, 3H), 0.97 (t, J = 7.5 Hz, 3H); m/z (ESI) 275 [M + H]⁺. 6-ethyl-7-oxo-5- (trifluoromethyl)-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹HNMR (300 MHz, CD₃OD) δ 8.08 (s, 1H), 2.63 (q, J = 7.2 Hz, 2H), 1.06 (t, J = 7.2 Hz, 3H); m/z (ESI) 257 [M + H]⁺. 6-allyl-5-methyl-7-oxo- 4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, DMSO) δ 13.18 1H), 8.37 (s, 1H), 5.83 (m, 1H), 5.02 (m, 2H), 3.29 (m, 2H), 2.35 (s, 3H); m/z (ESI) 215 [M + H]⁺. 6-isobutyl-5-methyl-7- oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, DMSO-d6) δ 13.16 (brs, 1H), 8.30 (s, 1H), 2.39-2.34 (5H), 1.86 (m, 1H), 0.85 (d, J = 6.6 Hz, 6H); m/z (ESI) 231 [M + H]. 6-ethyl-5-(furan-3-yl)- 7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹HNMR (300 MHz, CD₃OD) δ 8.28 (s, 1H), 8.05 (dd, J = 1.8 Hz, 0.9 Hz, 1H), 7.79 (m, 1H), 6.84 (dd, J = 1.8 Hz, 0.9 Hz, 1H), 2.66 (q, J = 7.5 Hz, 2H), 1.20 (t, J = 7.2 Hz, 3H); m/z (ESI) 277 [M + Na]⁺. 7-oxo-5- (trifluoromethyl)-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹HNMR (300 MHz, CD₃OD) δ 8.36 (s, 1H), 6.53 (s, 1H); m/z (ESI) 251 [M + Na]⁺. 6-cyclopropyl-5- methyl-7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹HNMR (300 MHz, CD₃OD) δ 8.19 (s, 1H), 2.59 (s, 3H), 1.33 (m, 1H), 1.01 (m, 2H), 0.73 (m, 2); m/z (ESI) 237 [M + Na]⁺.

Example 10 Synthesis of 4-methyl-9-oxo-4,5,6,7,8,9-hexahydropyrazolo[5,1-b]quinazoline-3-carbonitrile

9-oxo-4,5,6,7,8,9-hexahydropyrazolo[5,1-b]quinazoline-3-carbonitrile (50 mg, 0.23 mmol) was dissolved in DMF (1 mL), potassium carbonate (63 mg, 0.46 mmol) was added followed by iodomethane (36 mg, 0.26 mmol). The mixture was stirred at room temperature overnight, then diluted with water (10 mL) and extracted with EtOAc (5 mL×3). The combined organic layer was dried and concentrated to dryness. The residue was recrystallized from methanol to afford 12-3 (25 mg, 47%). ¹H NMR (300 MHz, CDCl₃) δ 8.07 (s, 1H), 3.94 (s, 3H), 2.69 (m, 4H), 1.92 (m, 2H), 1.76 (m, 2H); m/z (ESI) 229 [M+H]⁺.

By a similar method to Example 10, using the appropriate starting material, 6-ethyl-4,5-dimethyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile was prepared and isolated.

Compound Name Structure Data 6-ethyl-4,5- dimethyl-7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, CDCl₃) δ 8.09 (s, 1H), 4.00 (s, 3H), 2.71 (q, J = 7.5 Hz, 2H), 2.48 (s, 3H), 1.14 (t, J = 7.5 Hz, 3H); m/z (ESI) 217 [M + H]⁺.

Example 11 Synthesis of 4-(2-bromoethyl)-6-ethyl-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile

To a solution of tetrabutylammoniunbromide (557 mg, 1.73 mmol) in water (20 mL), NaOH (76 mg, 1.9 mmol) was added at ambient temperature, followed by 6-ethyl-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (350 mg, 1.73 mmol) in CHCl₃ (20 mL), the mixture continued to stir for 10 min. The organic phase was separated, the aqueous phase was extracted with CHCl₃ (10 mL). The combined organic phase was dried over anhydrous Na₂SO₄, evaporated to give white solid. The solid was dissolved in CH₃CN (10 mL), followed by 1,2-dibromoethane (360 mg, 2 mmol). The mixture continued to stir for 16 h at refluxing temperature, followed by concentration and purification with column chromatography to give 4-(2-bromoethyl)-6-ethyl-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (200 mg, 40%). m/z (ESI) 309 [M+H]⁺.

Example 12 Synthesis of 4-(2-(dimethylamino)ethyl)-6-ethyl-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile

To a solution of 4-(2-bromoethyl)-6-ethyl-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (200 mg, 0.65 mmol) in DMF (10 mL) were added potassium carbonate (446 mg, 3.24 mmol) and dimethylamine hydrochloride (155 mg, 1.94 mmol) consequently. The mixture was stirred at room temperature overnight, then diluted with water (10 mL) and extracted with EtOAc (10 mL×3). The combined organic layer was dried with Na₂SO₄, concentrated and purified by column chromatography to afford 4-(2-(dimethylamino)ethyl)-6-ethyl-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (12 mg, 10%). ¹H NMR (300 MHz, CD₃OD) δ 8.29 (s, 1H), 4.52 (t, J=7.2 Hz, 2H), 2.83 (t, J=7.2 Hz, 2H), 2.72 (q, J=7.5 Hz, 2H), 2.61 (s, 3H), 2.39 (s, 6H) 1.15 (t, J=7.5 Hz, 3H); m/z (ESI) 274 [M+H]⁺.

Example 13 Synthesis of 9-methoxy-5,6,7,8-tetrahydropyrazolo[5,1-b]quinazoline-3-carbonitrile

Synthesis of 9-chloro-5,6,7,8-tetrahydropyrazolo[5,1-b]quinazoline-3-carbonitrile

To a solution of 9-oxo-4,5,6,7,8,9-hexahydropyrazolo[5,1-b]quinazoline-3-carbonitrile (1.0 g, 4.6 mmol) in dry POCl₃ (20 mL) was added pyridine (0.2 mL) under a nitrogen atmosphere, the mixture was heated to 110° C. overnight. After cooling down to room temperature, the solvent was removed in vacuo, and the residue was purified by silica gel column chromatography to yield the desired compound 9-chloro-5,6,7,8-tetrahydropyrazolo[5,1-b]quinazoline-3-carbonitrile (0.6 g, yield 56%). ¹H NMR (300 MHz, CDCl₃) δ 8.36 (s, 1H), 3.10 (m, 2H), 2.93 (m, 2H), 1.96 (m, 4H).

Synthesis of 9-methoxy-5,6,7,8-tetrahydropyrazolo[5,1-b]quinazoline-3-carbonitrile

A mixture of 9-chloro-5,6,7,8-tetrahydropyrazolo[5,1-b]quinazoline-3-carbonitrile (100 mg, 0.43 mmol) and MeONa (48 mg, 0.86 mmol) in MeOH (4 mL) was stirred at room temperature for 1.5 hour, and the reaction was quenched by saturated NH₄Cl, and extracted with DCM (50 mL×3). The organic layer was washed by brine, dried over Na₂SO₄, concentrated in vacuo, and crude product was purified by preparation thick layer chromatography to yield the desired compound 9-methoxy-5,6,7,8-tetrahydropyrazolo[5,1-b]quinazoline-3-carbonitrile (20 mg, yield 21%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.25 (s, 1H), 4.44 (s, 3H), 3.02 (t, J=6.3 Hz, 2H), 2.80 (t, J=6.3 Hz, 2H), 1.89 (m, 4H); m/z (ESI) 229 [M+H]⁺.

By a similar method to Example 14, using the appropriate starting material, 6-ethyl-7-methoxy-5-methylpyrazolo[1,5-a]pyrimidine-3-carbonitrile was prepared and isolated.

Compound Name Structure Data 6-ethyl-7-methoxy- 5- methylpyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, CDCl₃) δ 8.28 (s, 1H), 4.43 (s, 3H), 2.77 (q, J = 7.8 Hz, 2H), 2.70 (s, 3H), 1.23 (t, J = 7.5 Hz, 3H); m/z (ESI) 217 [M]⁺

Example 14 Synthesis of 9-(2-hydroxyethoxy)-5,6,7,8-tetrahydropyrazolo[5,1-b]quinazoline-3 carbonitrile

A mixture of 9-chloro-5,6,7,8-tetrahydropyrazolo[5,1-b]quinazoline-3-carbonitrile (100 mg, 0.43 mmol), Et₃N (0.7 mL) in ethane-1,2-diol (2 mL) was stirred at room temperature overnight. The reaction was diluted with EtOAc (2.0 mL). The addition of hexane (2 mL) led to the precipitation of the product. The product was filtered, washed with EtOAc to yield the desired compound 9-(2-hydroxyethoxy)-5,6,7,8-tetrahydropyrazolo[5,1-b]quinazoline-3 carbonitrile (2.0 mg, yield 2%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.69 (s, 1H), 4.94 (t, J=5.1 Hz, 1H), 4.79 (t, J=4.5 Hz, 2H), 3.73 (m, 2H), 2.94 (t, J=5.7 Hz, 2H), 2.82 (t, J=6.3 Hz, 2H), 1.83 (m, 4H); m/z (ESI) 259 [M+H]⁺.

Example 15 Synthesis of 9-(2-methoxyethoxy)-5,6,7,8-tetrahydropyrazolo[5,1-b]quinazoline-3-carbonitrile

A mixture of 9-chloro-5,6,7,8-tetrahydropyrazolo[5,1-b]quinazoline-3-carbonitrile (100 mg, 0.43 mmol), Et₃N (0.7 mL) in 2-methoxyethanol (2 mL) was stirred at 70° C. overnight. The solvent was removed in vacuo, and the crude product was purified by preparation thin layer chromatography to yield the desired compound 9-(2-methoxyethoxy)-5,6,7,8-tetrahydropyrazolo[5,1-b]quinazoline-3-carbonitrile (10.0 mg, yield 9%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.24 (s, 1H), 4.92 (m, 2H), 3.75 (m, 2H), 3.37 (s, 3H), 3.03 (t, J=6.0 Hz, 2H), 2.84 (t, J=6.0 Hz, 2H), 1.91 (m, 41-1); m/z (ESI) 273 [M+H]⁺.

Example 16 Synthesis of 3-cyano-6-ethyl-N-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-5-carboxamide

Synthesis of 3-cyano-6-ethyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-5-carboxylic acid

To a solution of ethyl 3-cyano-6-ethyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-5-carboxylate (500 mg, 1.92 mmol) in EtOH (50 mL) and H₂O (5 mL) at 0° C., LiOH (230 mg, 9.6 mmol) was added in portions. The reaction mixture was stirred at room temperature for 2 h. After removal of EtOH in vacuo, water (5 mL) was added, and the mixture was filtered to produce 3-cyano-6-ethyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-5-carboxylic acid (380 mg, 89%) as off-white solid. ¹H NMR (300 MHz, DMSO-d6) δ 8.41 (s, 1H), 2.67 (q, J=6.9 Hz, 2H), 1.09 (t, J=6.9 Hz, 3H).

Synthesis of 3-cyano-6-ethyl-N-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-5-carboxamide

To a solution of 3-cyano-6-ethyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-5-carboxylic acid (50 mg, 0.21 mmol), methanamine hydrochloride (15 mg, 0.21 mmol), PyBOP (105.20 mg, 0.21 mmol) and HOBT (41.85 mg, 0.31 mmol) in DMF (2 mL) was added DIEA (129 mg, 1 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with CH₂Cl₂ and purified by column chromatography to produce 3-cyano-6-ethyl-N-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-5-carboxamide (16 mg, 31%) as off-white solid. ¹HNMR (300 MHz, CD₃OD) δ 8.16 (s, 1H), 2.95 (s, 3H), 2.79 (q, J=7.5 Hz, 2H), 1.20 (t, J=7.5 Hz, 3H); m/z (ESI) 246 [M+H]⁺.

By a similar method as above, using the ethanamine hydrochloride as reagent, 3-cyano-N,6-diethyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-5-carboxamide was prepared and isolated.

Compound Name Structure Data 3-cyano-N,6-diethyl-7-oxo- 4,7-dihydropyrazolo[1,5- a]pyrimidine-5-carboxamide

¹H NMR (300 MHz, CD₃OD) δ 8.16 (s, 1H), 3.43 (q, J = 7.2 Hz, 2H), 2.77 (q, J = 7.5 Hz, 2H), 1.31 (t, J = 7.5 Hz, 3H), 1.21 (t, J = 7.2 Hz, 3H); m/z (ESI) 260 [M + H]⁺.

Example 17 Synthesis of 4-benzyl-9-oxo-4,5,6,7,8,9-hexahydropyrazolo[5,1-b]quinazoline-3-carbonitrile

To solution of 9-oxo-4,5,6,7,8,9-hexahydropyrazolo[5,1-b]quinazoline-3-carbonitrile (50 mg, 0.23 mmol) in DMF (1 mL), potassium carbonate (63 mg, 0.46 mmol) and sodium iodide (5 mg, 3 mmol) were added followed by benzyl bromide (43 mg, 0.25 mmol). The mixture was stirred at room temperature overnight, then diluted with water (10 mL) and extracted with EtOAc (5 mL×3). The combined organic layer was dried and concentrated to dryness. The residue was purified by thick layer chromatography on silica gel to afford 4-benzyl-9-oxo-4,5,6,7,8,9-hexahydropyrazolo[5,1-b]quinazoline-3-carbonitrile (25 mg, 35%). ¹H NMR (300 MHz, CDCl₃) δ 8.03 (s, 1H), 7.38 (m, 3H), 7.04 (d, J=5.6 Hz, 2H), 5.56 (s, 2H), 2.68 (m, 4H), 1.79 (m, 4H); m/z (ESI) 305 [M+H]⁺.

Example 18 Synthesis of 9-oxo-4-phenyl-4,5,6,7,8,9-hexahydropyrazolo[5,1-b]quinazoline-3-carbonitrile

To a solution of 9-oxo-4,5,6,7,8,9-hexahydropyrazolo[5,1-b]quinazoline-3-carbonitrile (645 mg, 3 mmol) in CH₂Cl₂ (20 mL) was added Cu(OAc)₂, 4 A molecular sieve, Et₃N (607 mg, 6 mmol) and pyridine (474 mg, 6 mmol) consequently, the solution was stirred for 2 days under O₂. To the mixture was added NH₃H₂O and adjusted to PH>8. After filtration, the solution was concentrated and purified by column chromatography to give 9-oxo-4-phenyl-4,5,6,7,8,9-hexahydropyrazolo[5,1-b]quinazoline-3-carbonitrile (60 mg, 10%) as white solid. ¹H NMR (300 MHz, CD₃Cl) δ 7.99 (s, 1H), 7.52-7.36 (5H), 2.81 (m, 2H), 2.52 (m, 2H), 1.80 (m, 4H); m/z (ESI) 291 [M+H]⁺.

Example 19 Synthesis of 6-ethyl-5-(hydroxymethyl)-2-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile

To a solution of 6-ethyl-5-(methoxymethyl)-2-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (50 mg, 0.2 mmol.) in CH₂Cl₂ (20 mL) at −78° C., BCl₃ (1 mL, 1 mmol) was added dropwise, the mixture continued to stir at −78° C. for 2 h, then allowed to room temperature for 16 h. The mixture was quenched by water, and the aqueous phase was washed by CH₂Cl₂ (20 mL×3). After removal of water, the residue was washed with CH₂Cl₂/MeOH (50 mL, V/V=20/1) and filtered. The solution was concentrated and purified by column chromatography to give 6-ethyl-5-(hydroxymethyl)-2-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (10 mg, 23% yield) as off-white solid. ¹H NMR (300 MHz, CD₃OD) δ 4.71 (s, 2H), 2.61 (m, 2H), 2.49 (s, 3H), 1.56 (t, J=7.2 Hz, 3H); m/z (ESI) 233 [M+H]⁺.

By a similar method as above, using the appropriate starting materials, the compounds in Table 5 were prepared and isolated.

TABLE 5 Compound Name Structure Data 6-ethyl-5- (hydroxymethyl)-7- oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, CD₃OD) δ 8.24 (s, 1H), 4.73 (s, 2H), 2.64 (m, 2H), 1.17 (t, J = 7.5 Hz, 3H); m/z (ESI) 219 [M + H]⁺ 5-(hydroxymethyl)- 7-oxo-6-propyl-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, CD₃OD) δ 8.30 (s, 1H), 4.78 (s, 2H), 2.63 (m, 2H), 1.64 (m, 2H), 1.06 (t, J = 7.5 Hz, 3H); m/z (ESI) 233 [M + H]⁺ 6-(2-hydroxyethyl)- 5-methyl-7-oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, DMSO-d6) δ 13.08 (s, 1H), 8.35 (s, 1H), 4.61 (brs, 1H), 3.50 (m, 2H), 2.63 (t, J = 6.6 Hz, 2H), 2.39 (s, 3H); m/z (ESI) 219 [M + H]⁺. 6-ethyl-5-(3- hydroxypropyl)-7- oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹H NMR (300 MHz, CD₃OD) δ 8.22 (s, 1H), 3.70 (t, J = 6.0 Hz, 2H), 2.86 (m, 2H), 2. 65 (q, J = 7.5 Hz, 2H), 1.93 (m, 2H), 1.19 (t, J = 7.5 Hz, 3H); m/z (ESI) 247 [M + H]⁺ 6-allyl-5- (hydroxymethyl)-7- oxo-4,7- dihydropyrazolo[1,5- a]pyrimidine-3- carbonitrile

¹HNMR (300 MHz, CD₃OD) δ 8.26 (s, 1H), 5.94 (m, 1H), 5.08 (m, 2H), 4.71 (s, 2H), 3.41 (d, J = 6.0 Hz, 2H); m/z (ESI) 253 [M + Na]⁺.

Example 20 Synthesis of 6-ethyl-5-oxo-5,7,8,9-tetrahydropyrazolo[1,5-a]pyrrolo[1,2-c]pyrimidine-1-carbonitrile

To a solution of 6-ethyl-4,7-dihydro-5-(3-hydroxypropyl)-7-oxopyrazolo[1,5-a]pyrimidine-3-carbonitrile (12 mg, 0.05 mmol) in THF (10 mL) was added DIAD (10 mg, 0.06 mmol) and PPh₃ (16 mg, 0.06 mmol) at 0° C. The mixture was allowed to room temperature and stirred for overnight. The mixture was evaporated in vacuo, purified by column chromatography to give 6-ethyl-5-oxo-5,7,8,9-tetrahydropyrazolo[1,5-a]pyrrolo[1,2-c]pyrimidine-1-carbonitrile (5 mg, 43.8%). ¹H NMR (300 MHz, CD₃OD) δ 8.24 (s, 1H), 4.55 (t, J=7.5 Hz, 2H), 3.23 (t, J=7.8 Hz, 2H), 2.60 (q, J=7.5 Hz, 2H), 2.47 (m, 2H), 1.18 (t, J=7.5 Hz, 3H); m/z (ESI) 229 [M+H]⁺.

Example 21 Synthesis of 6-ethyl-5-formyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile

To a solution of DMSO (780 mg, 5 mmol) in CH₂Cl₂ (20 mL) was added dropwise oxalyl dichloride (127 mg, 5 mmol) at −78° C., then continued to stir for 30 min. 6-ethyl-5-(hydroxymethyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (110 mg, 0.5 mmol) in CH₂Cl₂ (5 mL) was added dropwise. After stirring for 1 h, Et₃N (1.5 g, 15 mmol) was added dropwise and allowed to ambient temperature. The mixture was quenched by water, extracted with CH₂Cl₂ (10 mL×3), combined organic layer was dried over anhydrous Na₂SO₄ and evaporated. The crude was purified by column chromatography to give 6-ethyl-5-formyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (50 mg, 50%) as yellow solid. ¹H NMR (300 MHz, CD₃Cl) δ 10.12 (s, 1H), 8.32 (s, 1H), 3.04 (q, J=7.2 Hz, 2H), 1.23 (t, J=7.2 Hz, 3H); m/z (ESI) 217 [M+H]⁺.

Example 22 Synthesis of 6-ethyl-5-(1-hydroxyethyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile

To a solution of 6-ethyl-5-formyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (50 mg, 0.23 mmol) in THF (10 mL) at −60° C., CH₃MgBr (0.7 mL, 0.68 mmol) was added dropwise, and continued to stir for 1 h. The mixture was quenched by saturated NH₄Cl, and extracted by CH₂Cl₂ (20 mL×5). Combined organic layer was dried over anhydrous Na₂SO₄ and evaporated. The residue was purified by column chromatography to give 6-ethyl-5-(1-hydroxyethyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (5 mg, 15%) as white solid. ¹H NMR (300 MHz, CD₃OD) δ 8.29 (s, 1H), 5.22 (m, 1H), 2.65 (m, 2H), 1.59 (d, J=6.6 Hz, 3H), 1.24 (t, J=7.5 Hz, 3H); m/z 233 [M+H]⁺.

Example 23 Synthesis of 9-oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-3-carbonitrile

Synthesis of 2-acetamidobenzoic acid

Anthranilic acid (100 g, 0.73 mol) in Ac₂O (1000 mL) was heated at 100° C. for 2 h. The Ac₂O was evaporated in vacuo and the residue was washed with hexane to give the product 2-acetamidobenzoic acid (130 g, 100%) as off-white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.19 (m, 1H), 7.79 (m, 1H), 7.54 (m, 2H), 2.48 (s, 3H).

Synthesis of 3-amino-2-methylquinazolin-4(3H)-one

A mixture of 2-acetamidobenzoic acid (6.0 g, 33.5 mmol) and NH₂NH₂.H₂O (5.9 g, 100 mmol) was stirred at 0° C. for 10 min and heated to reflux for 30 min. After removal of solvent, the residue was washed with ethanol to give 3-amino-2-methylquinazolin-4(3H)-one as off-white solid (1 g, 12.5%). ¹H NMR (300 MHz, CD₃OD) δ 8.24 (m, 1H), 7.75 (m, 2H), 7.46 (m, 1H), 2.72 (s, 3H).

Synthesis of 9-oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-3-carbaldehyde

The Vilsmeier-Haack reaction was performed according to Pandit, R. S.; Seshadri, S. Vilsmeier-Haack reaction. Indian. J. Chem. 1973, 11(6), 532-537. To a solution of POCl₃ (2.7 mL, 29 mmol) in DMF (5 mL), was added 3-amino-2-methylquinazolin-4(3H)-one (1.0 g, 5.7 mmol) in DMF (10 mL) at 0° C. Then the mixture was heated at 70° C. for 5 h and poured into crushed ice. The resulting creamy solution was basified with NaHCO₃ to pH=8 at 0° C. when a bright yellow crystalline compound separated out. It was filtered, washed with water. The solid was taken in potassium carbonate solution (10%, 10 mL) and warmed at 60° C. for half an hour when a clear yellow solution was obtained. The solution was neutralized to pH=5 with HCl (1N), and filtered to give 9-oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-3-carbaldehyde as off-white solid (800 mg, 82.5%). ¹H NMR (300 MHz, DMSO-d6) δ 9.90 (s, 1H), 8.44 (s, 1H), 8.24 (m, 1H), 7.86 (m, 2H), 7.42 (m, 1H).

Synthesis of 9-oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-3-carbaldehyde oxime

9-Oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-3-carbaldehyde (800 mg, 3.75 mmol) and hydroxylamine hydrochloride (250 mg, 3.6 mmol) were taken in EtOH (50 mL) and reflux for 3 h. The solvent was removed in vacuo to give the crude product 9-oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-3-carbaldehyde oxime (700 mg, 82%) as off-white solid. ¹H NMR (300 MHz, DMSO-d6) δ 12.48 (s, 1H), 11.35 (s, 1H), 8.57 (s, 1H), 8.21 (m, 1H), 7.82 (m, 1H), 7.52 (m, 1H), 7.34 (m, 1H).

Synthesis of 9-oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-3-carbonitrile

To a solution of 9-oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-3-carbaldehyde oxime (600 mg, 2.63 mmol) in dry CHCl₃ (10 mL) was added phosphorus oxychloride (0.5 mL, 5.5 mmol) and the mixture refluxed for 2 h. After removal of CH₃Cl, ice-cooled water was added followed by sodium bicarbonate to adjust to pH around 8. Precipitate was filtered, washed with water to give 9-oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-3-carbonitrile (200 mg, 30%) as off-white solid. ¹H NMR (300 MHz, DMSO-d6) δ 13.33 (s, 1H), 8.43 (s, 1H), 8.23 (d, J=8.1 Hz, 1H), 7.86 (dd, J=8.4 Hz, 6.9 Hz, 1H), 7.58 (d, J=8.1 Hz, 1H), 7.41 (dd, J=8.1 Hz, 7.2 Hz, 1H); m/z (ESI) 211 [M+H]⁺.

Example 24 Synthesis of 3-ethyl-2-methyl-4-oxo-3,4-dihydropyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile

Synthesis of (E)-ethyl N-4-cyano-1H-pyrazol-5-ylacetimidate

Analogous to chemistry described in U.S. Pat. No. 4,892,576. A solution of 5-amino-1H-pyrazole-4-carbonitrile (1.1 g, 10 mmol), 1,1,1-triethoxyethane (2 g, 12 mmol) and AcOH (3 drops) in MeCN (74 mL) was refluxed for 16 h. The resulting mixture was cooled down to room temperature, evaporated under vacuo. The residue was purified with column chromatography to yield (E)-ethyl N-4-cyano-1H-pyrazol-5-ylacetimidate (400 mg, 22%). ¹H NMR (300 MHz, CDCl₃) δ 7.80 (s, 1H), 4.33 (q, J=7.2 Hz, 2H), 2.10 (s, 3H), 1.38 (t, J=7.2 Hz, 3H).

Synthesis of 3-ethyl-2-methyl-4-oxo-3,4-dihydropyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile

To a solution of (E)-ethyl N-4-cyano-1H-pyrazol-5-ylacetimidate (100 mg, 0.56 mmol) in anhydrous THF (5 mL) was added TEA (57 mg, 0.56 mmol) and isocyanatoethane (50 mg, 0.7 mmol) at 0° C. The mixture was warmed to ambient temperature and stirred for 18 h. The solvent was removed at reduced pressure, and the residue was purified with column chromatography to yield 3-ethyl-2-methyl-4-oxo-3,4-dihydropyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile (10 mg, yield 8.8%). ¹H NMR (300 MHz, CD₃OD) δ 8.32 (s, 1H), 4.23 (q, J=7.2 Hz, 2H), 2.73 (s, 3H), 1.42 (t, J=7.2 Hz, 3H); m/z (ESI) 204 [M+H]⁺.

Example 25 Synthesis of 6-ethyl-5-((methylamino)methyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile

Synthesis of 5-(bromomethyl)-6-ethyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile

To a solution of 6-ethyl-5-(hydroxymethyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (50 mg, 0.228 mmol) and PPh₃ (120 mg, 0.456 mmol) in CH₂Cl₂ (10 mL) was added CBr₄ (152 mg, 0.456 mmol) at ambient temperature. The reaction mixture was stirred for 16 h. The residue was concentrated and purified by column chromatography to give 5-(bromomethyl)-6-ethyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (30 mg, 50%). m/z (ESI) 281/283 [M+H]⁺.

Synthesis of 6-ethyl-5-((methylamino)methyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile

To a solution of 5-(bromomethyl)-6-ethyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (30 mg, 0.11 mmol) in 5 ml of DMF, Et₃N (22 mg, 0.22 mmol) and methanamine hydrochloride (15 mg, 0.22 mmol) was added. The mixture was stirred at ambient temperature for 16 h. The residue was concentrated and purified by column chromatography to afford 6-ethyl-5-((methylamino)methyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (6 mg, 20%). ¹HNMR (300 MHz, CD₃OD) δ 8.18 (s, 1H), 4.34 (s, 2H), 2.89 (s, 3H), 2.66 (q, J=7.5 Hz, 2H), 1.20 (t, J=7.2 Hz, 3H); m/z (ESI) 232 [M+H]⁺.

Example 26 Assessment of Inhibitory Effect of Test Compounds on GASC1 Demethylase Activity on Histone 3 Lysine 9 Trimethyl Peptide (H3K9Me3) GASC1 Demethylation Assay

6×His tagged recombinant GASC1 (N 350aa) was purified from E. Coli BL21(DE3) to near homogeneity. The demethylation reaction buffer contained 50 mM TrisCl pH 7.5, 0.01% Triton X-100, 5% glycerol, 1 mM ascorbate (Cat# A4034, Sigma Aldrich), 5 μM α-ketoglutarate (# K2010, Sigma Aldrich) and 20 μM Fe₂(NH₄)₂(SO₄)₂ (Cat# F1543, Sigma Aldrich). In 25 μL demethylation reaction system, 400 nM recombinant GASC1 and 20 μM H3K9me3 peptide (1-21 aa) were incubated with compounds for 10 minutes, and then α-ketoglutarate and Fe₂(NH₄)₂(SO₄)₂ were added to initiate the reaction. All of the reactions were incubated for 45 minutes at room temperature, and then 25 μl of 1 N HCl was added to quench the reactions. After termination, plates were sealed and frozen at −80° C. and shipped on dry ice to BioTrove Inc. (Woburn, Mass.) for anaylsis.

High Throughput Mass Spectrometry (HT-MS) Analysis

All the reactions were read by RapidFire™ HT-MS platform developed in BioTrove Inc, and the method has been described in detail previously (Assay and Drug Development Technologies, 2004; 2(4): 373-381). Briefly, at BioTrove, plates were thawed and immediately analyzed using RapidFire™ system coupled to a Sciex API4000 triple quadrapole mass spectrometer. The samples were delivered directly from the plate to a clean-up cartridge (BioTrove column A) to remove nonvolatile assay components with 0.1% formic acid in a 3-sec wash cycle. The peptide substrate and demethylated product were coeluted to the mass spectrometer with 80% acetonitrile, 0.1% formic acid. Both the substrate and product signals were read at their +5 charge species, and the conversion from substrate to product is assessed by [H3K9me2 Read]/[H3K9me2 Read +H3K9me3 Read].

Example 27

Table 6 shows the activity of selected compounds of this invention in the GASC1 inhibition assay. The compound numbers correspond to the compound numbers in Table 1. Compounds having an activity designated as “A” provided an IC₅₀≦1 μM; compounds having an activity designated as “B” provided an IC₅₀ 1-10 μM; compounds having an activity designated as “C” provided an IC₅₀ of 10-50 μM; and compounds having an activity designated as “D” provided an IC₅₀≧50 μM.

TABLE 6 GASC1 Inhibition Data Compound # GASC1 Inhibition  I-1 D  I-2 D  I-3 B  I-4 A  I-5 B  I-6 D I-12 C I-13 D I-14 D I-15 B I-16 B I-17 A I-18 D I-19 D I-20 B I-21 A I-22 B I-23 B I-24 B I-25 A I-26 B I-27 D I-28 D I-29 D I-30 B I-31 B I-32 B I-33 B I-34 A I-35 B I-36 D I-37 D I-38 A I-39 B I-40 B I-41 A I-42 A I-43 B I-44 A I-45 A I-46 A I-47 A I-48 B I-49 B I-50 B I-51 A I-52 B I-53 B I-54 B I-55 B

Example 28 Assessment of Inhibitory Effect of Test Compounds on JARID1A and PLU-1 Demethylase Activity on Histone 3 Lysine 4 Trimethyl Peptide (H3K4Me3) JARID1A/PLU1 Demethylase Assays

FLAG tagged full length recombinant JARID1A and PLU1 proteins were purified from Sf9 insect cells to near homogeneity. The demethylation reaction buffer contained 50 mM TrisCl pH 7.5, 0.01% Triton X-100, 0.005% BSA, 1 mM ascorbate (Cat# A4034, Sigma Aldrich), 1.7 μM α-ketoglutarate (# K2010, Sigma Aldrich) and 20 μM Fe₂(NH₄)₂(SO₄)₂ (Cat# F1543, Sigma Aldrich). In a 25 μL demethylation reaction system, 20 nM recombinant JARID1A or PLU1 proteins and 4 μM H3K4me3 peptide (1-21 aa), which can be biotinylated or unlabelled, were incubated with compounds for 10 minutes, and then α-ketoglutarate and Fe₂(NH₄)₂(SO₄)₂ were added to initiate the reaction. All of the reactions were incubated for 45 minutes at room temperature, and then 25 μl of 1 N HCl was added to quench the reactions. After termination, plates were sealed and frozen at −80° C. for anaylsis.

High Throughput Mass Spectrometry (HT-MS) Analysis

All the reactions were read by RapidFire™ HT-MS platform developed in BioCius Inc, and described in detail (Assay and Drug Development Technologies, 2004; 2(4): 373-381). Briefly, plates were thawed and immediately analyzed using RapidFire™ system coupled to a Sciex API4000 triple quadrapole mass spectrometer. The samples were delivered directly from the plate to a clean-up cartridge (BioCius column A) to remove nonvolatile assay components with 0.1% formic acid in a 3-sec wash cycle. The peptide substrate and demethylated product were coeluted to the mass spectrometer with 80% acetonitrile, 0.1% formic acid. Both the substrate and product signals were read at their +5 charge species, and the conversion from substrate to product assessed by [H3K4me2 Read]/[H3K4me2 Read +H3K4me3 Read].

Example 29

Table 7 shows the activity of selected compounds of this invention in the JARID1A and PLU-1 inhibition assays. The compound numbers correspond to the compound numbers in Table 1. Compounds having an activity designated as “A” provided an IC₅₀≦1 μM; compounds having an activity designated as “B” provided an IC₅₀≦1-10 μM; compounds having an activity designated as “C” provided an IC₅₀ of 10-50 μM; and compounds having an activity designated as “D” provided an IC₅₀≧50 μM.

TABLE 7 JARID1A and PLU-1 Inhibition Data Compound # JARID1A PLU-1  I-4 B B I-21 A A I-23 A A I-25 A A I-29 B B I-30 A A I-49 A B

While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example. 

We claim:
 1. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; each R is independently hydrogen or an optionally substituted group selected from C₁₋₆ aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated, partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R′ is independently —R, —C(O)R, —CO₂R, or two R′ on the same nitrogen are taken together with their intervening atoms to form a 4-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Ring A is

R² and R³ are independently —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R² and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R^(2′) is —R, —OR, —SR, —N(R′)₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R^(2′) and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X is —N(R⁴)—, —O—, or —S—; R⁴ is —R, —C(O)R, —CO₂R, or —S(O)₂R; or: R⁴ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered saturated, partially unsaturated, or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R⁵ is R, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)C(O)R, or —C(O)CH₂C(O)R; or: R⁵ and R² are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and R⁶ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R⁶ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; provided that the compound is other than any one of the following:


2. The compound according to claim 1, wherein said compound is of formula II:

or a pharmaceutically acceptable salt thereof.
 3. The compound according to claim 2, wherein R⁴ is hydrogen.
 4. The compound according to claim 1, wherein R¹ is hydrogen.
 5. The compound according to claim 1, wherein R² is optionally substituted C₁₋₆ aliphatic.
 6. The compound according to claim 5, wherein R² is methyl, ethyl, propyl, cyclopropyl, isopropyl, isobutyl, propargyl, or allyl.
 7. The compound according to claim 1, wherein R³ is optionally substituted C₁₋₆ aliphatic.
 8. The compound according to claim 7, wherein the C₁₋₆ aliphatic group is substituted with —OH or —OC₁₋₆alkyl.
 9. The compound according to claim 7, wherein the C₁₋₆ aliphatic group is substituted with —NHC₁₋₆alkyl or —NH(C₁₋₆alkyl)₂.
 10. The compound according to claim 7, wherein R³ is optionally substituted benzyl.
 11. The compound according to claim 7, wherein R³ is one of the following:


12. The compound according to claim 7, wherein R³ is —CF₃.
 13. The compound according to claim 1, wherein R³ is —CO₂R or —C(O)N(R′)₂.
 14. The compound according to claim 1, wherein R³ is a 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
 15. The compound according to claim 1, wherein neither of R² and R³ is hydrogen.
 16. The compound according to claim 1, wherein said compound is of formula:

or a pharmaceutically acceptable salt thereof.
 17. The compound according to claim 16, wherein said compound is of formula:


18. A compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 19. A composition comprising a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; each R is independently hydrogen or an optionally substituted group selected from C₁ aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated, partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R′ is independently —R, —C(O)R, —CO₂R, or two R′ on the same nitrogen are taken together with the intervening nitrogen to form a 4-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Ring A is

R² and R³ are independently —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R² and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R^(2′) is —R, —OR, —SR, —N(R′)₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R^(2′) and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X is —N(R⁴)—, —O—, or —S—; R⁴ is —R, —C(O)R, —CO₂R, or —S(O)₂R; or: R⁴ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered saturated, partially unsaturated, or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R⁵ is R, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)C(O)R, or —C(O)CH₂C(O)R; or: R⁵ and R² are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R⁶ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R⁶ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a pharmaceutically acceptable adjuvant, carrier, or vehicle.
 20. A method for inhibiting activity of a 2-oxoglutarate dependent enzyme, or a mutant thereof, activity in a patient comprising the step of administering to said patient a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; each R is independently hydrogen or an optionally substituted group selected from C₁₋₆ aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated, partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R′ is independently —R, —C(O)R, —CO₂R, or two R′ on the same nitrogen are taken together with the intervening nitrogen to form a 4-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Ring A is

R² and R³ are independently —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R² and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R^(2′) is —R, —OR, —SR, —N(R′)₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R^(2′) and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X is —N(R⁴)—, —O—, or —S—; R⁴ is —R, —C(O)R, —CO₂R, or —S(O)₂R; or: R⁴ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered saturated, partially unsaturated, or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R⁵ is R, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)C(O)R, or —C(O)CH₂C(O)R; or: R⁵ and R² are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and R⁶ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R⁶ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or a composition according to claim
 19. 21. A method for treating a GASC1-mediated disorder in a patient in need thereof, comprising the step of administering to said patient a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′) C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; each R is independently hydrogen or an optionally substituted group selected from C₁₋₆ aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated, partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R′ is independently —R, —C(O)R, —CO₂R, or two R′ on the same nitrogen are taken together with the intervening nitrogen to form a 4-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Ring A is

R² and R³ are independently —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R² and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R^(2′) is —R, —OR, —SR, —N(R′)₂, —C(O)R, —CO₂R, —C(O)N(R¹)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R^(2′) and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X is —N(R⁴)—, —O—, or —S—; R⁴ is —R, —C(O)R, —CO₂R, or —S(O)₂R; or: R⁴ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered saturated, partially unsaturated, or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R⁵ is R, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)C(O)R, or —C(O)CH₂C(O)R; or: R⁵ and R² are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and R⁶ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R⁶ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. or a composition according to claim
 19. 22. The method of claim 20, wherein the 2-oxoglutarate dependent enzyme is a JARID family enzyme.
 23. A method for treating a JARID-mediated disorder in a patient in need thereof, comprising the step of administering to said patient a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; each R is independently hydrogen or an optionally substituted group selected from C₁ aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated, partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R′ is independently —R, —C(O)R, —CO₂R, or two R′ on the same nitrogen are taken together with the intervening nitrogen to form a 4-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Ring A is

R² and R³ are independently —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R² and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R^(2′) is —R, —OR, —SR, —N(R′)₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R¹)₂; or: R^(2′) and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X is —N(R⁴)—, —O—, or —S—; R⁴ is —R, —C(O)R, —CO₂R, or —S(O)₂R; or: R⁴ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered saturated, partially unsaturated, or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R⁵ is R, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)C(O)R, or —C(O)CH₂C(O)R; or: R⁵ and R² are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and R⁶ is —R, halogen, —OR, —SR, —N(R′)₂, —CN, —NO₂, —C(O)R, —CO₂R, —C(O)N(R′)₂, —C(O)SR, —C(O)C(O)R, —C(O)CH₂C(O)R, —C(S)N(R′)₂, —C(S)OR, —S(O)R, —SO₂R, —SO₂N(R′)₂, —N(R′)C(O)R, —N(R′)C(O)N(R′)₂, —N(R′)SO₂R, —N(R′)SO₂N(R′)₂, —N(R′)N(R′)₂, —N(R′)C(═N(R′))N(R′)₂, —C═NN(R′)₂, —C═NOR, —C(═N(R′))N(R′)₂, —OC(O)R, or —OC(O)N(R′)₂; or: R⁶ and R³ are taken together with their intervening atoms to form an optionally substituted 5-7 membered partially unsaturated or aromatic fused ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a composition according to claim
 19. 